Immunotherapy of cancer with allogeneic lymphocytes

ABSTRACT

Methods have been discovered for treating residual disease following removal of most or a substantial fraction of malignant cells from a cancer patient. An autologous stem cell transplant is performed on the patient. Following partial hematopoiesis recovery, the patient is infused with allogeneic peripheral blood lymphocytes, either alone or in combination with in vivo or in vitro T-cell activation. The infused allogeneic lymphocytes engender and anti-malignant cell response.

This application is a 371 of PCT/US95/03303, filed Mar. 16, 1995 and isa continuation in part of Ser. No. 08/214,944, filed Mar. 17, 1994.

FIELD OF THE INVENTION

This invention relates to methods of eradicating residual tumor cellsfollowing surgical intervention, chemotherapy and/or radiotherapy. Themethods involve administration of allogeneic lymphocytes followingautologous stem cell transplantation. More particularly, this inventionrelates to use of HLA-compatible or HLA-mismatched allogeneiclymphocytes to induce a graft-versus-malignant cell response followingautologous stem cell transplantation.

BACKGROUND OF THE INVENTION

Patients with malignant hematological disorders resistant toconventional doses of chemotherapy and/or radiation may be treated byautologous or allogeneic bone marrow transplantation. Bone marrowtransplantation (BMT) makes it possible to administer to patients withresistant disease high, "supralethal," combinations of chemotherapy andradiation, ignoring the irreversible toxicity of such therapeuticcombinations on the normal bone marrow compartment. Nevertheless, such"debulking" of a patient's tumor(s) can leave a fraction of residualmalignant cells that may lead to disease relapse. Several lines ofevidence suggest that a significant proportion of the beneficial effectof allogeneic BMT (i.e., BMT from an individual not geneticallyidentical to the host patient) stems from cell-mediated interactions ofimmune cells of donor origin against residual tumor cells in the hostthat have escaped the chemoradiotherapy debulking regimen.

Following allogeneic BMT, the incidence of relapse is significantlylower in leukemia patients with clinical manifestations of acute orchronic graft versus host disease (GVHD), as compared with patients withno GVHD, indicating that immune-mediated allogeneic interactions orimmunocompetent cells of donor origin against the host are alsoaccompanied by graft vs. leukemia (GVL) effects. Weiden et al., N. Engl.J. Med. 300: 1068 (1979); Weiden et al., N. Engl. J. Med. 304: 1529-33(1981); Weiden et al., Transplantation 13: 248-51 (1981); Barrett etal., Blood 74: 862 (1989); Sullivan et al., Blood 73:1720 (1989);Horowitz et al., Blood 75: 555 (1990); Slavin et al., Bone MarrowTransplant. 6: 155-61 (1990).

Higher relapse rates seem to occur in patients undergoing allogeneic BMTwith T-lymphocyte depletion for prevention of GVHD compared torecipients of non-T cell depleted marrow allografts, regardless of theseverity of GVHD. Horowitz et al., Blood 75:555 (1990); Slavin et al.,Bone Marrow Transplant. 6:155-61 (1990); Goldman et al., Ann. Inter.Med. 108: 806-14 (1988); Ringden and Horowitz, Transplant. Proc. 21:2989-92 (1989); Goldman et al., Ann. Int. Med. 108: 806 (1988).Likewise, relapse rates in patients with acute leukemia or chronicmyeloid leukemia reconstituted by bone marrow grafts obtained from anidentical twin (syngeneic grafts) are significantly higher than in thosereconstituted by bone marrow cells obtained from an HLA-identical butnon-syngeneic sibling. Ringden and Horowitz, Transplant. Proc. 21:2989-92 (1989). Similarly, relapse rates following transplantation ofthe patient's own (autologous) marrow, even following adequate purgingin vitro for elimination of residual leukemia cells, are significantlyhigher than following allogeneic BMT. Armitage, Curr. Opinion inHematol. 1993: 240-45 (1993). Thus, the less-than optimal results withautologous BMT (ABMT) are similar to the results seen with syngeneicmarrow transplantation. All of the above data suggests that in practicalterms GVHD or GVHD-potential correlates with a lower incidence ofrelapse.

Allogeneic donor cells may also play a role against lymphoma, as shownin experimental animals, Slavin et al., Cancer Immunol. Immunother. 11:155-58 (1981), and humans. Phillips et al., J. Clin. Oncol. 4: 480-88(1986); Ernst et al., Proc. of the 4th International Conference onLymphoma, Lugano 1990, Abstract #P35; Chopra et al., J. Clin. Oncol. 10:1690-95 (1992). As shown in experimental animals, graft-versus-tumoreffects (GVT), similar to graft versus-leukemia effects (GVL), may occurfollowing BMT, independently of GVHD. Moscovitch and Slavin, J. Immunol.132: 997-1000 (1984). As used herein, GVL is one form of GVT.

Although GVHD-associated anti-leukemia effector mechanisms may be ofbenefit in BMT, nevertheless GVHD represents one of the major obstaclesin allogeneic BMT, even among perfectly HLA-matched siblings. Acute GVHDdevelops in the majority of recipients of allogeneic BMT, withclinically significant manifestations in 26-64% of the recipientsdespite optimal post-transplant immunosuppressive prophylaxis. Storb etal., Blood 73: 1729 (1989). Chronic GVHD may occur in up to 45% of longterm survivors. Storb et al., Blood 73: 1729 (1989). There is nosatisfactory therapy for patients with established GVHD and hencepatients with severe manifestations of acute or chronic forms of thedisease are prone to develop multisystem complications that may requirefrequent hospitalizations, leading to poor quality of life andoccasionally serious or even fatal complications.

GVHD following allogeneic BMT can be prevented by adequate pretransplantT-lymphocyte depletion, using no post-transplant anti-GVHD prophylaxis.Reisner et al., In: Slavin, S(ed.), Tolerance in Bone Marrow and OrganTransplantation. Elsevier, Amsterdam (1984), p. 293; Waldmann et al.,Lancet 2: 483-85 (1984); Slavin et al., Transplant. Proc. 17: 465-67(1985). BMT without GVHD represents a better tolerated procedure thatmay necessitate shorter hospitalization with superior subjectiveimmediate outcome following allogeneic BMT. In addition, the quality oflife of long-term survivors without GVHD is clearly better than forthose patients with severe acute or chronic GVHD.

Unfortunately, the advantages of GVHD-free-allogeneic BMT arecounterbalanced by other serious complications due to untoward effectsof T-lymphocyte depletion, such as increased incidence of graftrejection, occurring in 10-30% of recipients, as well as increased ratesof tumor relapse. Martin et al., Bone Marrow Transplant. 3: 445 (1988);Kernan et al., Transplantation 43: 842 (1987); Waldmann et al., Lancet2: 483-85 (1984); Slavin et al., Transplant. Proc. 17: 465-67 (1985).Consequently, there seems to be no clear evidence to date for asignificant overall benefit of GVHD prevention by T-lymphocytedepletion.

Clearly, it would be a significant advance in the art to be able tocombine the benefits of minimal or controllable GVHD risk following ABMTor autologous stem cell transplantation (ASCT) with induction ofgraft-versus-malignant cell response that may be associated with GVHDfollowing allogeneic BMT.

SUMMARY OF THE INVENTION

The present invention includes a method of treating a human cancerpatient who has undergone a malignant cell debulking procedure in whichautologous stem cell transplantation has been performed incident to thedebulking procedure. In other words, the patient's own stem cells wereinfused back into the patient in order to reconstitute the patient'sbone marrow following the tumor debulking. Generally the patient isconsidered to be at risk for disease relapse due to a population ofresidual malignant cells that may remain viable in the patient followingthe debulking procedure. The patient is monitored until the patient ispartially hematopoiesis recovered but is not fully immune-reconstituted.Then, the patient is administered allogeneic lymphocytes in a regimenthat causes a clinically significant graft-versus-malignant cellresponse. Allogeneic lymphocytes in this setting are lymphocytes takenfrom an individual not genetically identical to the patient into whichthe lymphocytes are infused. The patient is monitored for levels ofmalignant cells deriving from any residual malignant cells that mighthave been present following the original debulking procedure. Thismonitoring may constitute one or more molecular or cellular assays todetect or quantify malignant cells, may constitute a monitoring programto detect clinical signs of relapse, or any combination of thesemonitoring methods.

At used herein, a clinically significant response permits, for example,the patient to avoid relapse, substantially prolongs the time to relapseor otherwise engenders a beneficial condition that significantlyprolongs life. Thus, evidence for a clinically significant response mayinclude, for example, absence or delay of relapse, induction oftemporary or permanent remission, evidence for elimination of minimalresidual disease, i.e., elimination of disease-specific markers and,where appropriate, elimination of markers directed to host-specificcells.

In situations where allogeneic lymphocytes are not selected (see below)to reduce or eliminate the GVHD potential of the cells, it is preferablethat HLA-compatible allogeneic lymphocytes are used. A regimen utilizingHLA-compatible allogeneic lymphocytes may comprise the following stepsin sequence:

a) treating the patient by administration (e.g., infusion) of about 10⁷cells/kg to about 10⁹ cells/kg of HLA-compatible lymphocytes;

b) monitoring the patient for indications of a graft-versus-malignantcell response; and

c) if no or insufficient graft-versus-malignant cell response developsin the patient, escalating the treatment by performing at least one ofthe following procedures: (1) administration of a number ofHLA-compatible, allogeneic lymphocytes greater than the number oflymphocytes administered in step (a); (2) administration of a number ofHLA-compatible, allogeneic lymphocytes at least as great as the numberof lymphocytes administered in step (a), accompanied by in vivoadministration of at least one T-cell activator to the patient; (3)administration of HLA-compatible, allogeneic activated donor lymphocytes(ADL) to the patient; and (4) administration of HLA-compatible,allogeneic ADL, accompanied by administration in vivo of at least oneT-cell activator to the patient. More than one of these procedures canbe performed if no or insufficient graft-versus-malignant cell responsedevelops in the patient following the first or subsequent procedure.

In an alternative embodiment, step (a) above can be augmented byadministering, concomitant with the allogeneic lymphocytes, at least oneT-cell activator to the patient. Since the T-cell activator isadministered directly to the patient, the infused allogeneic lymphocytesare exposed to the activator in vivo.

The present invention also includes an alternative method of treating ahuman cancer patient in which GVHD is used as a clinical marker.Following malignant cell debulking and autologous stem celltransplantation, the patient is monitored until the patient is partiallyhematopoiesis recovered but is not fully immune-reconstituted. Then, thepatient is administered HLA-compatible, allogeneic lymphocytes in aregimen that causes a mild graft-versus-host response. The patient isthen monitored, as above, for levels of malignant cells deriving fromany residual malignant cells that might have been present following theoriginal debulking procedure.

As used herein, the term "graft-versus-host response" includes but isnot limited to the classic clinical symptoms of graft-versus hostdisease (GVHD), known to those having ordinary skill in the art.Patients with a mild graft-versus-host response include those with, forexample, Grade I or Grade I/II cutaneous GVHD or other forms of GVHDthat stop short of severe manifestations leading to serious or fatalmultisystem complications. A mild graft-versus-host response could alsoinclude molecular or cellular responses that correlate with the clinicalsymptoms of GVHD or with the impending onset of the clinical symptoms ofGVHD.

For the above-described alternative method involving mild GVHD, theregimen for administration of HLA-compatible lymphocytes may comprisethe following steps in sequence:

a) treating the patient by administration of about 10⁷ cells/kg to about10⁹ cells/kg of HLA-compatible, allogeneic lymphocytes;

b) monitoring the patient for indications of a mild graft-versus-hostresponse; and

c) if no or insufficient graft-versus-host response develops in thepatient, escalating the treatment by performing at least one of thefollowing procedures: (1) administration of a number of HLA-compatible,allogeneic lymphocytes greater than the number of lymphocytesadministered in step (a); (2) administration of a number ofHLA-compatible, allogeneic lymphocytes at least as great as the numberof lymphocytes administered in step (a), accompanied by in vivoadministration of at least one T-cell activator to the patient; (3)administration of HLA-compatible, allogeneic ADL to said patient; and(4) administration of HLA-compatible, allogeneic ADL, accompanied byadministration in vivo of at least one T-cell activator to the patient.More than one of these procedures can be performed if no or insufficientgraft-versus-host response develops in the patient following the firstor subsequent procedure.

In an alternative embodiment, step (a) above can be augmented byadministering, concomitant with the allogeneic lymphocytes, at least oneT-cell activator to the patient. Since the T-cell activator isadministered directly to the patient, the infused allogeneic lymphocytesare exposed to the activator in vivo.

In a further alternative embodiment involving mild GVHD, the regimen foradministration of HLA-compatible lymphocytes may comprise the followingsteps in sequence:

a) administering to the patient about 10⁷ cells/kg to about 10⁹ cells/kgof HLA-compatible, allogeneic lymphocytes and at least one T-cellactivator to the patient;

b) monitoring the patient for signs of a mild graft-versus-hostresponse;

c) if no or insufficient graft-versus-host response develops in thepatient, administering about 10⁷ cells/kg to about 10⁹ cells/kg ofHLA-compatible, allogeneic ADL and at least one T-cell activator to thepatient;

d) monitoring the patient for signs of a mild graft-versus-hostresponse;

Alternatively, the ADL may be given in the initial infusion. In thiscase the regimen for administration of HLA-compatible lymphocytes maycomprise the following steps in sequence:

a) administering to the patient of about 10⁵ cells/kg to about 10⁹cells/kg of HLA-compatible, allogeneic lymphocytes in which at leastsome of the HLA-compatible, allogeneic lymphocytes are ADL, togetherwith a T-cell activator, to the patient;

b) monitoring the patient for signs of a mild graft-versus-hostresponse;

c) if no or insufficient graft-versus-host response develops in thepatient, administering about 10⁵ cells/kg to about 10⁹ cells/kg ofHLA-compatible, allogeneic ADL and at least one T-cell-activator to thepatient;

d) monitoring the patient for signs of mild graft-versus-host response;

In any of the methods described herein, the T-cell activator can be anysuitable agent that activates the T-cell signal transduction pathwayleading to lymphocyte activation. Lymphocyte activation involves aseries of interrelated events described, for example, in Abbas et al.,Cellular and Molecular Immunology, Second Edition, W. B. Saunders Co.(1994), pages 153-65. A T-cell activator may comprise, withoutlimitation, any one or more of the following: interleukin 1 (IL1),interleukin 2 (IL2), interleukin 4 (IL4), interleukin 5 (IL5),interleukin 6 (IL6), interleukin 7 (IL7), interleukin 12 (IL12),interleukin 13 (IL13), interferon alpha (IFNα), interferon gamma (IFNγ),tumor necrosis factor (TNFα), an anti-CD3 antibody or antigen-bindingfragments thereof (anti-CD3), an anti-CD28 antibody or antigen-bindingfragments thereof (anti-CD28), phytohemagglutinin, concanavalin-A andphorbol esters. Any of these activators can be a native factor obtainedfrom natural sources, a factor produced by recombinant DNA methodology,a chemically synthesized polypeptide or other molecule, or anyderivative having the functional activity of the native factor.

The stem cells used for autologous stem cell transplantation may beobtained from bone marrow, from the peripheral circulation, or, whereappropriate, from fetal sources such as fetal tissue, fetal circulationand umbilical cord blood. Cancer patients treatable with the methods ofthe present invention are any patients having a pathological conditioncaused by malignant cells, including without limitation leukemia,lymphoma, and breast cancer.

In an alternative embodiment, the allogeneic lymphocytes administered tothe patient in the various methods of the present invention may beselected to have a substantially diminished graft-versus-host activitycompared to unselected lymphocytes. Preferably the selected lymphocytesare CD8+ lymphocytes. In cases where selected lymphocytes havingsubstantially diminished graft-versus-host activity are used, thelymphocytes may be either HLA compatible or HLA-mismatched with thepatient. In an initial infusion of HLA-mismatched lymphocytes, forexample HLA-mismatched CD8+lymphocytes, as few as about 10⁵ to as manyas about 10⁹ cells may be administered.

The present invention also includes the use of a T-cell activator in themanufacture of a medicament for the treatment of a human cancer patient,where the patient has undergone a malignant cell debulking procedure andhas further undergone autologous stem cell transplantation incident tothe debulking procedure. As above, the patient is at risk for diseaserelapse due to a population of residual malignant cells that may remainviable in the patient following the debulking procedure. The treatmentof this patient involves one or more of the Allo-CMI and/or Allo-CCImethods of the invention described above.

The invention further includes the use of allogeneic lymphocytes (i.e.,allogeneic to a patient to be treated) in the manufacture of amedicament for the treatment of a human cancer patient, where thepatient has undergone a malignant cell debulking procedure and hasfurther undergone autologous stem cell transplantation incident to thedebulking procedure. As above, the patient is at risk for diseaserelapse due to a population of residual malignant cells that may remainviable in the patient following the debulking procedure. The treatmentof this patient involves one or more of the Allo-CMI and/or Allo-CCImethods of the invention described above.

The present invention further includes an article of manufacturecomprising packaging material and a container within the packagingmaterial. The packaging material contains a label or package insertindicating that the contents of the packaging material may be used inany of the above-described methods of the invention for treating a humancancer patient. Preferably a container included in the packagingmaterial is a collapsible container (e.g., a plastic bag) comprisingopposing walls of flexible material and a flexible tube (e.g., a plastictube) protruding from the container. A T-cell activator may be containedwithin the container. Preferably the tube is adapted to receiveallogeneic lymphocytes (i.e., allogeneic to a patient to be treated)into the container. If T-cell activator is present in the container, thein-coming lymphocytes become activated. These ADL then can be used totreat a cancer patient according to the methods of the inventiondescribed above.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts the results of adoptive transfer of spleen cells obtainedfrom lethally irradiated F1 mice transplanted with 10⁷ syngeneic bonemarrow cells and 10⁵ BCL1 cells in addition to 20-30×10⁶ PBL fromallogeneic mice with (A, n=40) or without (B, n=40) concomitant in vivorhIL2 treatment (12×10⁴ IU×2/day for 5 days IP). A similar groupreceived syngeneic (F1) PBL given with (C, n=20) or without (D, n=20)rhIL2. A control group had adoptive transfer to spleen cells obtainedfrom 10 untreated F1 recipients (E, n=20).

FIG. 2 depicts the results of adoptive transfer of spleen cells obtainedfrom lethally irradiated F1 mice reconstituted with 10⁷ syngeneic bonemarrow cells mixed with 10⁵ BCL1 cells. Cell-mediated immunotherapyconsisted of intravenous administration of increasing numbers of C57BL/6spleen cells: 1×10⁶ (A, n=10); 3×10⁶ (B, n=10); 10×10⁶ (C, n=10) and30×10⁶ (D, n=10). One of three experiments is shown.

FIG. 3 depicts results of adoptive transfer of spleen cells obtainedfrom BALB/c mice treated with cyclophosphamide (300 mg/kg IP),inoculated 24 hours later with 10³ BCL1 cells, and one day laterreceiving 4×10⁶ syngeneic, ASTAZ-treated bone marrow cells.Immunotherapy consisted of a mixture of 20×10⁶ allogeneic C57BL/6 spleenand lymph node cells either with (A, n=6) or without (B, n=6) rhIL2treatment in vivo (12×10⁴ IU×3/day for 3 days IP). Recipients of amixture of 20×10⁶ syngeneic BALB/c spleen and lymph node cells treatedwith rhIL2 (C, n=7) and recipients of 10³ BCL1 cells only (D, n=10)served as controls.

FIG. 4 depicts the results of adoptive transfer of spleen cells obtainedfrom lethally irradiated F1 mice inoculated with 10⁵ BCL1 cells and30×10⁶ bone marrow cells pre-activated in vitro for 4 days with rhIL2;mice with no additional treatment (A, n=33), mice with in vivo rhIL2treatment (12×10⁴ IU×2/day for 5 days IP) (B, n=25); controls:recipients of 10⁵ spleen cells obtained from untreated control miceinoculated with 10⁵ BCL1 cells (C, n=30).

FIG. 5 shows photographs of computerized tomography (CT) scans of abreast cancer patient. Panel A shows the presence of breast cancermetastases (arrows) in the liver before Allo-CMI/CCI. The CT image inPanel B fails to reveal liver metastases in the same patient after beinggiven an Allo-CMI/CCI regimen.

FIG. 6 depicts the results of adoptive transfer of spleen cells obtainedfrom irradiated F1 mice inoculated with 10⁵ BCL1 cells. The irradiatedmice received unselected allogeneic T cells or T-cell populationsselected for CD4+ or CD8+ cells; control mice received syngeneic (F1) Tlymphocytes. A =unselected C57 spleen cells; B =CD8+ cells, i.e., spleencells treated with an antibody that depletes the cell population of CD4+cells; C =CD4+ cells, i.e., spleen cells treated with an antibody thatdepletes the cell population of CD8+ cells; D=unselected control (F1)spleen cells.

FIG. 7 depicts the results of adoptive transfer of spleen cells obtainedfrom irradiated F1 mice inoculated with 10⁵ BCL1 cells. The irradiatedmice received unselected allogeneic T cells or T-cell populationsselected for CD4+ or CD8+ cells; control mice received syngeneic (F1) Tlymphocytes. The cells were administered as ADL in concert with in vivoadministration of rIL2. A =unselected C57 ADL administered withoutconcomitant in vivo rIL2; B=unselected C57 ADL plus in vivo rIL2; C=CD8+ADL, i.e., ADL treated with an antibody that depletes the cellpopulation of CD4+ cells, plus in vivo rIL2; D=CD4+ ADL, i.e., ADLtreated with an antibody that depletes the cell population of CD8+cells, plus in vivo rIL2.

DETAILED DESCRIPTION OF THE INVENTION

The present inventor has employed allogeneic peripheral bloodlymphocytes, alone or in combination with T-cell activator treatment invivo or in vitro, for successful elimination of minimal residual diseasefollowing chemotherapy and/or radiotherapy. Appropriate treatmentregimens have been circumscribed by studies in laboratory animals, andthe treatment protocols have been further extended to human patients athigh risk for disease relapse.

A spontaneous, transplantable murine B-cell leukemia/lymphoma of BALBorigin (BCL1) was used to investigate elimination of minimal residualdisease (MRD) following bone marrow transplantation. As used herein, MRDrefers to a condition wherein residual malignant cells remain viable ina patient following a primary and/or metastatic tumor "debulking"procedure. The debulking procedure may comprise any protocol thatremoves or destroys tumor cells, including without limitation surgicalexcision, chemotherapy or radiotherapy, or any combination of theseapproaches. Such treatment removes a significant fraction of malignantcells from the patient, but may leave a clinically significant number ofresidual malignant cells that put the patient at risk of relapse. Theterm "tumor" as used herein includes all pathological conditionsinvolving malignant cells; this can include "solid" tumors arising insolid tissues or organs as well as "liquid" tumors such as leukemias andlymphomas deriving from malignant transformation of hematopoietic cells.

In autologous bone marrow transplantation (ABMT) with human patients, anindividual receives his or her own bone marrow cells by infusionfollowing a tumor debulking procedure. Generally, the bone marrow cellsare taken from the patient and preserved, for example bycryopreservation, prior to the debulking procedure. ABMT permits anotherwise lethal debulking regimen to be employed, e.g., chemotherapy orradiotherapy that severely damages or destroys the patient's bonemarrow. Following the debulking procedure, the patient's bone marrow isreconstituted by stem cells present in the preserved sample of bonemarrow.

Stem cells capable of reconstituting a patient's immune system can beobtained not only by direct extraction from the bone marrow, but alsofrom the patient's peripheral circulation following mobilization of suchcells from the bone marrow. This can be accomplished by treatment of thepatient with granulocyte colony stimulating factor (G-CSF) or otherappropriate factors that induce movement of stem cells from the bonemarrow into the peripheral circulation. Following mobilization, the stemcells can be collected from peripheral blood by any appropriate cellapheresis technique, for example through use of a commercially availableblood cell collection device as exemplified by the CS 3000® Plus bloodcell collection device marketed by the Fenwal Division of BaxterHealthcare Corporation. Methods for performing apheresis with the CS3000® Plus machine are described in Williams et al., Bone MarrowTransplantation 5: 129-33 (1990) and Hillyer et al., Transfusion 33:316-21 (1993), both publications being incorporated herein by reference.Stem cells collected from the peripheral blood are termed herein"peripheral blood stem cells" (PBSC). The term "autologous stem celltransplantation" (ASCT) is used herein to refer to any infusion into apatient of that same patient's stem cells, derived from any appropriatesource (e.g., bone marrow or peripheral circulation). As such, ABMT,where the autologous infused cells are extracted directly from the bonemarrow of the patient, may be considered simply one form of ASCT.

It is possible to create an experimental regime in mice that simulatesASCT in humans. This is done through use of stem cell donors andrecipients derived from a syngeneic strain of mice. In such strains,inbreeding has created a situation in which, for practical purposes,mice within the strain are genetically identical to each other. Suchmice accept tissues and organs transplanted between individuals withoutevidence of immune rejection, in a manner analogous to acceptance of apatient's own cells following ASCT. Transplantation of bonemarrow-derived stem cells between such mice is referred to herein as"syngeneic bone marrow transplantation" (SBMT) and may be consideredanalogous to ABMT and ASCT in humans.

In the present experiments, mice received a lethal dose of total bodyirradiation (TBI) or, alternatively, a lethal dose of cyclophosphamideadministered intraperitoneally. Bone marrow cells (BMC) were extracteddirectly from the bone marrow of syngeneic mice. In some cases, theseBMC preparations were treated with mafosfamide (ASTA-Z) to simulate a"purging" procedure in which the patient's stem cells, prior to ASCT,are treated to remove or destroy at least a fraction of anycontaminating malignant cells. One day after irradiation or treatmentwith cyclophosphamide, the mice received 10⁷ syngeneic bone marrow cellsby infusion into the lateral tail vein. To simulate MRD, 10⁵ BCL1 tumorcells were added to the syngeneic BMC prior to SBMT.

Following SBMT, recipient mice received either allogeneic cell-mediatedimmunotherapy (Allo-CMI) or allogeneic cell-mediated cytokine-activatedimmunotherapy (Allo-CCI). Allo-CMI involved transfer of immunocompetentallogeneic lymphocytes (i.e., lymphocytes from a mouse strain other thanthat of the recipient mice) at various times and at various doses,administered post-SBMT. Generally these lymphocytes representedperipheral blood lymphocytes (PBL) or mixtures of donor spleen and lymphnode cells. Allo-CCI involved transfer of allogeneic lymphocytespre-activated in vitro with recombinant human interleukin 2 (rhIL2). Asused herein, the term "ADL" refers to such "activated donorlymphocytes," i.e., lymphocytes (human or mouse) activated in vitro witha T-cell activator such as rhIL2. In some experimental protocols, theAllo-CMI or Allo-CCI regimen was accompanied by simultaneous in vivoadministration of rhIL2 to the recipient mice, in order to facilitateadditional activation of the infused lymphocytes in vivo.

Failure to develop leukemia in primary recipients does not proveelimination of all BCL1 cells, since active suppression of existingtumor cells can prevent development of overt disease in these animals.This has been documented following allogeneic BMT and rhIL2 therapy.Slavin et al., Cancer Immunol. Immunother. 11:155 (1981); Slavin et al.,Nat. Immun. Cell Growth Regul. 7:180 (1988). To establish conclusiveevidence for eradication of residual malignant cells, spleen cells fromthe treated, or recipient, mice were adoptively transferred to secondarysyngeneic recipients. If these secondary recipients failed to developleukemia, it was judged that the original Allo-CMI- or Allo-CCI-treatedmice were free of viable malignant cells, since as few as 1-10 cellshave been shown to be capable of causing disease. Slavin et al., CancerRes. 41:4162 (1981); Cohen et al., J. Immunol. 151: 4803-10 (1993).

The results of the SBMT experiments with mice suggested that effectiveimmunotherapy of MRD can be achieved in vivo by cell therapy withalloreactive lymphocytes through an effect that can be further enhancedin vivo with a short course of intermediate-dose rhIL2. GVL-like effectswere also induced by infusion of ADL without causing any grossimpairment of the hematopoietic capacity of BMC in lethally irradiatedrecipients. Moreover, GVL effects induced by allogeneic lymphocytes aswell as ADL could be further enhanced by concomitant rhIL2 therapy invivo, most likely due to continuous in vivo activation of allogeneiceffector cells against residual host malignant cells.

The data further indicate that eradication of BCL1 can be accomplishedbefore overt clinical manifestations of GVHD in the primary recipientswould have occurred, since experiments showed that GVL-like effectsagainst BCL1 cells were achieved within 1-2 weeks followingadministration of allogeneic lymphocytes. This implies that temporaryengraftment of allogeneic effector cells may be sufficient to inducebeneficial GVL effects against MRD, without the need for permanentresidence of allogeneic effector cells, which may put the patient atrisk for severe GVHD across major histocompatibility barriers. Moreover,as the time interval between ASCT and the Allo-CMI/CCI treatmentincreases, larger numbers of donor's PBL can be administered with lesslikelihood of severe GVHD. Slavin et al., J. Exp. Med. 147:963 (1978);Slavin et al., Blood 80:535a (1992).

As such, it is preferred that an Allo-CMI or Allo-CCI regimen is begunonly after the patient is partially hematopoieses recovered followingthe original tumor debulking procedure/ASCT. This raises the likelihoodthat the allogeneic inoculum will be rejected by reconstituting hostimmune cells in due time, after GVT (e.g., GVL) effects have beenaccomplished. On the other hand, it is also preferred that theAllo-CMI/Allo-CCI regimen is undertaken prior to full immunereconstitution of the patient, since the likelihood of prematurerejection of the allogeneic inoculum, prior to beneficial GVL/GVTeffects, is thereby reduced. Since full immune reconstitution followingASCT in humans frequently requires up to one year, the patient can bemonitored until a stable clinical condition (e.g., condition ofstabilized blood counts) is attained, as indicated by acceptable levelsof, for example, white blood cells (WBC), hemoglobin (Hb) and platelets.This condition of partial hematopoieses recovery may be achievedbeginning in a matter of weeks following ASCT in humans, well beforefull immune reconstitution diminishes the likelihood of successfulAllo-CMI and/or Allo-CCI.

The Allo-CMI and Allo-CCI strategies developed in mice have been adaptedto human patients in a variety of protocols undertaken by the presentinventor in treating cancer patients at high risk of disease relapse.Cancer patients having acute myelogenous leukemia, chronic myelogenousleukemia, non-Hodgkin's lymphoma and metastatic breast cancer have beentreated with the methods of the present invention.

Two of the earlier-treated patients with acute myelogenous leukemia weregiven relatively low numbers (e.g., about 10⁴ cell/kg) of allogenicperipheral blood lymphocytes as a first dose, in an effort to minimizethe risk of serious GVHD. Thereafter, the patients were given escalatingnumbers of allogeneic peripheral blood lymphocytes at various timeintervals to increase the chances of clinically significant graft-versusmalignant cell response. Both of these early patients (patients 1 and 2in Example 2, set out below) relapsed and died. Following this result,it was apparent that administration of graded increments of PBL,beginning with relatively low numbers, may not be effective.

In hindsight, it can be hypothesized that the initial low dose ofallogeneic PBL produced only immunization without significant GVHD orgraft-versus-malignant cell response. Thus, later, higher doses ofallogeneic PBL, which otherwise might be effective, may have beenpromptly rejected by the patient and rendered ineffectual forgraft-versus-malignant cell activity. Therefore, in subsequent cases,more cells (e.g., at least about 10⁷ cells/kg) were administered in theinitial dose. With this initial higher dose of allogeneic cellsfollowing ASCT, satisfactory results have been obtained in many patientswithin the treatment population to date.

If human leukocyte antigen (HLA)-compatible allogeneic lymphocytes areused in the present procedures, such cells preferably are fullyHLA-matched with the patient. Alternatively, HLA-compatible allogeneiccells may be at least haploidentical with the patient. Thus, if theallogeneic cells are derived from a sibling of the patient, somemismatch may be tolerated. For example, the HLA-compatible allogeneiccells may, in some cases, be single HLA locus-mismatched as demonstratedby Patient No. 12 described in EXAMPLE 2 below. If the allogeneic cellsare derived from an unrelated individual, preferably the cells are fullyHLA matched with the patient.

In a preferred embodiment of the present invention, an initial dose ofat least about 10⁷ HLA-compatible allogeneic lymphocytes/kg is givenonce the patient has achieved a clinically stable condition (e.g.,stabilized blood counts), i.e., once the patient is partiallyhematopoiesis recovered. Preferably, the patient is also administeredT-cell activator in vivo, concomitant with administration of theHLA-compatible lymphocytes. Preferably the T-cell activator is humaninterleukin 2 produced through recombinant DNA technology (rhIL2) at adose of about 6×10⁶ IU of rhIL2/m² /day, by subcutaneous injection,beginning on the same day as infusion of the allogeneic lymphocytes.Other appropriate T-cell signal transduction activators as set out abovemay be used, either with or without rhIL2, as long as the desiredactivation in vitro is obtained.

If GVHD fails to develop in a patient given the above-described numbersof HLA-compatible allogeneic lymphocytes with concomitant in vivo T-cellactivator, then the treatment regimen is escalated. Preferably this isdone by administering a second dose of allogeneic HLA-compatiblelymphocytes preactivated in vitro with T-cell activator. Preferably theT-cell activator is a cytokine such as rhIL2, although, again, otherT-cell signal transduction activators may be used either with or withoutrHIL2.

Prior to administration of the second dose of cells comprising ADL, andcontingent on the particular status of an individual patient,cyclophosphamide (Cytoxan) or other appropriate immunosuppressants maybe administered to the patient to avoid rejection of the second dose ofcells. That is, the immunosuppressant is given in a dose effective forkilling or inactivating host T cells that might otherwise operate toreject the second allogeneic inoculum; the immunosuppressant may havethe added benefit of eliminating potential host suppressor cellfunctions that can interfere with the GVT effects of the infused ADL.Preferably, in vivo T-cell activator is administered to the patientconcomitant with the second dose of allogeneic cells.

Although the above-described preferred embodiment is presentlyrecommended, it is to be understood that any combination of allogeneiclymphocytes, in vivo T-cell activator and/or ADL is covered by thepresent invention, so long as the initial dose of cells corresponds to anumber of HLA-compatible allogeneic lymphocytes that elicits a hostresponse beyond mere immunization to a second dose of cells from thesame or similar donor. Generally, this initial dose is at least about10⁷ allogeneic peripheral blood lymphocytes/kg. However, it is possiblethat a lower dose of cells, e.g., about 10⁵ cells/kg, could be used if,for example, ADL were used in the initial infusion.

Between the Allo-CMI/CCI treatments or at the conclusion of anAllo-CMI/CCI regimen, the patient may be monitored for levels ofmalignant cells, i.e., for evidence of minimal residual disease. Suchmonitoring may comprise patient follow-up for clinical signs of relapse.The monitoring may also include, where appropriate, various molecular orcellular assays to detect or quantify any residual malignant cells. Forexample, in cases of sex-mismatched donors and recipients, residualhost-derived cells may be detected through use of appropriate sexmarkers such as Y chromosome-specific nucleic acid primers or probes. Incases of single HLA locus mismatches between donors and recipients,residual host cells may be documented by polymerase chain reaction (PCR)analysis of Class I or Class II loci that differ between the donor andrecipient. Alternatively, appropriate molecular markers specific fortumor cells can be employed. For example, nucleic acid primers and/orprobes specific for the bcr/abl translocation in chronic myelogenousleukemia, for other oncogenes active in various tumors, for inactivatedtumor suppressor genes, other tumor-specific genes, or any other assayreagents known to be specific for tumor cells, may be employed. Any ofthese or functionally comparable procedures may be used to monitor thepatient for evidence or residual malignant cells.

Under normal circumstances, recipients of autologous or allogeneic bonemarrow transplants receive only irradiated blood products when suchproducts are required by the patient. These products are irradiated inorder to avoid the possibility of engraftment of immunocompetentT-lymphocytes derived from the donor's blood product (e.g., platelets orred blood cells). In most institutions, irradiated blood products arealso used for patients receiving high dose conventional chemotherapywithout transplant, e.g., blood products given following induction ofremission in leukemia and lymphoma patients. obviously, the chances forengraftment of immunocompetent T-lymphocytes from otherwise mismatchedblood products is relatively small under normal circumstances. However,if immunosuppression is sufficient to permit engraftment, GVHD can be"stormy" and lethal.

In one embodiment of the present invention, described above,non-irradiated donor-type lymphocytes are used intentionally forinduction of graft-versus-malignant cell effects. The method isstructured to produce transient engraftment, so as to inducegraft-versus-malignant cell effects that may be accompanied by mildGVHD. Since the donor cells used in this embodiment are HLA-compatiblewith the recipient, chances of engraftment are better than if thedonor's cells were not functionally matched with the patient's majorhistocompatibility complex. Moreover, the chances of immediate rejectionon the one hand and lethal GVHD on the other hand are relatively smallbecause of the HLA compatibility. As such, the Allo-CMI/CCI protocols ofthis embodiment provide the possibility for transient engraftment ofdonor's PBL with effective GVT and with a minimal chance for inductionof severe GVHD.

In alternative embodiments, T-cell subsets may be selected that retainGVT activity but that have a reduced or absent capability for inducingGVHD. In these embodiments, the use of HLA-compatible allogeneiclymphocytes is not required. Thus, although it may be desirable in somecircumstances to used HLA-compatible lymphocytes, in other cases it ispermissible to have a mismatch at two or more loci (HLA-mismatchedlymphocytes). This is because the use of T-cell subsets with limitedGVHD potential allows GHVD to be minimized even if the infusedlymphocytes are not HLA compatible with the patient. In fact, GVTpotential is enhanced through use of HLA mismatched allogeneiclymphocytes.

For selection of an appropriate T-cell subset, the present inventor hasestablished that CD8+ T lymphocytes represent the effector cells of theGVT response. Experiments establishing this finding are set out inEXAMPLE 4 below. The ability of CD8+ cells to induce GVHD is reducedcompared to unselected T cells. As such, CD8+ cells represent a usefulT-cell subset for use in the clinical setting, since the risk of GVHD isdiminished while significant GVT activity is retained. The term"selected" as used herein means use of any procedure that provides aT-cell population relatively enriched for a desired T-cell subset. Thiscould include, for example, positive selection for CD8+ cells, orelimination or reduction of CD4+ cells leaving a populationcorrespondingly enriched for CD8+ cells compared to an unselectedpopulation.

The invention will be further understood with reference to the followingillustrative embodiments, which are purely exemplary, and should not betaken as limiting the true scope of the present invention as describedin the claims.

EXAMPLE 1 Syngeneic BMT (SBMT) in Mice Followed by Allo-CMI or Allo-CCI

I. METHODS

A. Mice

BALB/c (BALB), C57BL/6 (C57), and (BALB/c x C57BL/6)F1 (F1) mice, 2-6months old, were purchased from the breeding colony of the HebrewUniversity-Hadassah Medical School, Jerusalem. Mice were kept understandard conditions, with acidic water (ph 2.7) and no specialprotective measures. Mice were given 0.5% neomycin sulfate in theirdrinking water for 2 weeks post-transplantation.

B. Murine B-Cell Leukemia (BCL1)

BLC1, a spontaneous, transplantable B-cell leukemia/lymphoma of BALBorigin is characterized by marked (up to 50 fold) splenomegaly,accompanied by extreme peripheral blood lymphocytosis (>200,00/mm³) andresults in death of all mice inoculated with ≧10-100 tumor cells. Slavinet al., Nature 272:624 (1978); Slavin et al., Cancer Res. 41:4162(1981). BCL1 was maintained in vivo in BALB mice by IV passage of 10⁶-10⁷ peripheral blood lymphocytes (PBL) obtained from tumor bearingmice. Mice with marked lymphocytosis in the blood were subsequently usedas BCL1 cell donors for experimental mice. PBL counts for allexperimental groups were carried out weekly. Leukemia was defined as PBLcounts exceeding 20,000/mm³. At the peak of disease, PBL counts usuallyreached >100,000/mm³.

C. Mafosfamide (ASTA-Z)

ASTA-Z was kindly provided by Drs M. Peukert and H. Sindermann(Astawerke, Bielefeld, Germany) as a lyophilized powder and was freshlydissolved in saline before use. ASTA-Z has been employed in vitro toreduce or eliminate malignant cell populations from bone marrowpreparations. Douay et al., CR Acad.Sci.Paris t. 301, Ser III, no. 6:303(1985).

D. Conditioning with Radiation and Cyclophosphamide Prior to BMT

Mice were exposed to a single dose of 750 cGy total body irradiation(TBI) from a Philips X-ray unit (250 kV 20 mA) with a focus to skindistance of 70 cm at a dose rate of 60 cGy/min. Alternatively, mice wereconditioned with freshly dissolved cyclophosphamide (CY) (300 mg/kg)(Taro, Israel) given intraperitoneally (IP). Twenty-four hours later,mice received 10⁷ syngeneic marrow cells via the lateral tail vein.

E. Preparation of Bone Marrow Cells (BMC)

BMC were obtained from the femora, tibiae and humeri of syngeneic mice.Mononuclear cells containing 10⁷ BMC in 0.25 ml Hank's medium wereinjected into the lateral tail vein of recipients 24 hourspost-radiation.

F. Purging Procedure

Cells were resuspended at a concentration of 20×10⁶ cells/ml in Hank'smedium containing 4% human serum albumin. ASTA-Z was then added to afinal concentration of 100 μg/ml. Both untreated control cells andASTA-Z treated BMC were incubated at 37° C. for 30 minutes, washed twicein Hank's medium and counted. Purged or unpurged BMC (4×10⁶) wereinjected into BALB mice conditioned with CY.

G. Recombinant Human Interleukin-2 (rhIL2)

rhIL2 provided as 1 mg Proleukin (3×10⁶ Cetus Units, equivalent to18×10⁶ International Units) was kindly supplied by Dr. S. L. Aukerman,Cetus/Chiron, Calif., USA. rhIL2 was initially diluted with water forinjection and subsequently rediluted with dextrose 5%. Internationalunits (IU) are used throughout the remainder of the present application.

H. Activation of BMC by rhIL2

BMC were cultured in 225 cm³ flasks (Corning 25160-225, Corning Glass,Corning N.Y.) in RPMI 1640 medium (Beit Haemek, Israel) containingL-glutamine, non-essential amino acids, pyruvate, 10% bovine calf serum(BCS) and rhIL2 (6,000 IU/ml) for 4 days in a humidified incubator with5% CO₂ at 37° C. Following harvesting, viability was determined by thetrypan blue exclusion method.

I. Simulation of Minimal Residual Disease Following Syngeneic BoneMarrow Transplantation

In order to simulate minimal residual disease (MRD) quantitatively, 10⁵BCL1 cells were added to the marrow inoculum during syngeneic bonemarrow transplantation (SBMT), prior to immunotherapy.

J. Immunotherapy by Immunocompetent Allogeneic Lymphocytes

Allogeneic cell-mediated immunotherapy (Allo-CMI) consisted of adoptivetransfer of immunocompetent allogeneic lymphocytes (PBL or a mixture ofdonor spleen and lymph node cells) as detailed in the results for eachexperiment, below. Allogeneic cell-mediated cytokine-activatedimmunotherapy (Allo-CCI) consisted of adoptive transfer of allogeneiclymphocytes pre-activated in vitro with T-cell activator (activateddonor lymphocytes, or "ADL"). In this EXAMPLE, T-cell activatorcomprised rhIL2. In some experiments allogeneic lymphocyte infusion wasfollowed by subsequent in vivo activation with rhIL2, by additionalAllo-CMI with in vivo rhIL-2, or by additional Allo-CCI with in vivorhIL-2, respectively.

K. Detection of Residual Clonogenic BCL1 by Adoptive TransferExperiments

In order to determine whether or not residual BCL1 cells were presentafter various treatments, 10⁵ spleen cells obtained from treated micewere adoptively transferred to untreated secondary syngeneic (BALB)recipients. Absence of leukemia (≧100 days) in secondary recipients wasindicative of elimination of BCL1 since as few as 1-10 cells werepreviously shown to cause disease.

L. Statistical Analysis

The significance of differences between treated and untreated mice wascalculated by the independent statistical t-test.

II. RESULTS

A. Induction of Allo-CMI and Allo-CCI Effects

F1 mice were lethally irradiated (750 cGy) and transplanted with 10⁷syngeneic BMC. Following inoculation of 10⁵ BCL1 cells to simulate MRD,varying numbers of C57 PBL were administered intravenously to induceGVL-like effects through allo-CMI. In order to detect the efficacy ofallo-CMI in eradicating residual BCL1 cells, aliquots of 10⁵ spleencells pooled from 2-3 experimental mice were adoptively transferred tosecondary normal BALB recipients, one or two weeks post-SBMT.

FIG. 1 summarizes results obtained from three different experiments in atotal of 120 mice. Injection of 20-30×10⁶ PBL, obtained from C57 mice toinduce allo-CMI after SBMT in F1 recipients, effectively eliminatedresidual BCL1 cells, as none of 40 secondary adoptive BALB recipientsdeveloped leukemia (>180 days). In contrast, leukemia developed in all20 secondary BALB recipients inoculated with 10⁵ spleen cells obtainedfrom F1 recipients that had received 20-30×10⁶ PBL from syngeneic donorspost-SBMT. Addition of rhIL2 (12×10⁴ IU×2/day for 5 days IP)post-transplant did not improve the disease-free survival of secondaryrecipients of 10⁵ spleen cells (obtained from similarly treated F1 mice)since all 20 secondary recipient BALB mice developed leukemia (FIG. 1).Addition of rhIL2 in vivo at the same dose to recipients of 20×10⁶allogeneic PBL for further in vivo activation of effector cells did notinduce measurable additional GVL effects since all 40 secondary BALBrecipients remained disease free (>180 days) (FIG. 1).

B. quantitative Effect of the Number of Effector Cells on the Efficacyof Allo-CMI

Anti-leukemic effects mediated by allo-CMI were cell-dose dependent. Asshown in FIG. 2, all SBMT recipients injected with 30×⁶ C57 spleen cellscompletely resisted the development of leukemia following inoculation of10⁵ BCL1 cells. Injection of 10×10⁶ allogeneic spleen cells togetherwith 10⁵ BCL1 cells induced effective allo-CMI in 70% of the secondaryadoptive recipients. However, reduction of allo-CMI inducing C57 spleencells to 3×⁶ or 1×⁶ failed to eliminate residual BCL1 cells and allsecondary adoptive recipients developed leukemia (FIG. 2).

C. Induction of Allo-CMI and Allo-CMI/Il-2 Effects FollowingTransplantation with ASTA-Z-Purged BMC

The feasibility of induction of allo-CMI was investigated byconditioning with high-dose CY followed by rescue of recipients withASTA-Z-purged syngeneic BMC. BALB recipients received high-dose CY (300mg/kg IP) and were injected 24 hours later with 10³ BCL1 cells tosimulate MRD. One day later, all mice received intravenously 4×⁶ ASTA-Ztreated syngeneic BMC. Mice were divided into 3 experimental groups: thefirst group (6 mice) received intravenously a mixture of allogeneic C57spleen and lymph node cells (20×⁶ cells) for induction of allo-CMI; thesecond group (6 mice) received identical cell therapy with additional invivo potentiation of GVL by rhIL2 treatment (12×⁴ IU×3/day for 3 days,IP); the third group (7 mice) received a mixture of syngeneic spleen andlymph node cells, with an identical in vivo rhIL2 treatment. One weeklater, aliquots of 10⁵ cells from a pool of 2-3 spleens obtained fromeach experimental group were adoptively transferred to secondary BALBmice.

As shown in FIG. 3, all mice inoculated with spleen cells from controlmice given 10³ BCL1 cells, or mice given syngeneic BALB lymphocytes within vivo rhIL2, developed leukemia and died within 40 and 60 days,respectively. Likewise, secondary recipients of 10⁵ spleen cellsobtained from mice that were treated with allo-CMI, using allogeneic C57cells alone, showed no measurable GVL effects since all recipientsdeveloped leukemia. In contrast, addition of rhIL2 in vivo followingadministration of C57 spleen and lymph node cell mixtures inducedsubstantial anti-leukemic effects and 50% of the secondary adoptiverecipient mice remained leukemia-free for >125 days (FIG. 3).

D. Enhancement of Immunotherapeutic Effect by In Vitro Activation ofAllogeneic Lymphocytes with rhIL2

The following experiment was designed to test for potential enhancementsin efficacy of treatment by in vitro pre-activation of allogeneiceffector cells with rhIL2. Lethally irradiated (750 cGy TBI) F1 micewere infused with 30×⁶ C57 BMC pre-activated in vitro for 4 days withrhIL2. BMC were mixed with 10⁵ BCL1 cells to simulate MRD. Results of 3separate sets of experiments gave similar results and therefore the datawere pooled (FIG. 4).

All F1 recipients were divided into two groups. The first group of 33mice received no additional treatment. Mice in the second group (25mice) were injected with rhIL2 (12×⁴ IU×2/day for 5 days, IP) in anattempt to further increase efficacy of cell therapy by continuousactivation of rhIL2-dependent effector cells in vivo. Aliquots of 10⁵cells obtained from a pool of spleen cells prepared from mice of eitherexperimental group were adoptively transferred to secondary BALBrecipients. As shown in FIG. 4, 10 of 33 secondary recipients of spleencells obtained from the first experimental group remained disease-freefor >150 days. Additional in vivo rhIL2 therapy in the secondexperimental group further improved the Allo-CCI effects, as 19 of 25secondary recipients remained disease-free for an observationperiod >150 days (p=0.05) (FIG. 4).

EXAMPLE 2 Autologous Stem Cell Transplantation (ASCT) in Humans Followedby Allo-CMI and/or Allo-CCI

I. PATIENT TREATMENT PROTOCOLS

Patient No. 1

This female patient was diagnosed with acute myelogenous leukemia (AML),French American British (FAB) classification M4, and was in firstcomplete remission (i.e., no evidence of disease) at the time ofautologous stem cell transplantation (ASCT). The patient was 41 yearsold at the time of ASCT. Prior to ASCT, she received a conditioningregimen of Busulfan, 4 mg/kg, days 6 through 9 pre-ASCT (days -9 to -6),as well as Cytoxan (cyclophosphamide), 50 mg/kg, days -5 to -2,Cotrimoxazol, 10 mg/kg, days -10 to -2, Allopurinol, 300 mg/kg, days -10to -1 and cytosine arabinoside (Ara-C), 25 mg intrathecally.

Prior to ASCT, the autologous cells to be infused were purged bytreatment with Mafosfamide (ASTA-Z). ASTA-Z was provided by Drs. M.Peukert and H. Sindermann (Astawerke, Bielefeld, Germany) as alyophilized powder and was freshly dissolved in saline before use.Autologous cells were resuspended at a concentration of 20×10⁶ cells/mlin Hank's medium containing 4% human serum albumin. ASTA-Z was thenadded to a final concentration of 100 ug/ml and the cells were incubatedin the ASTA-Z at 37° C. for 30 min. After this, the cells were washedtwice in Hank's medium and counted. Cells were cryopreserved and kept inliquid nitrogen until used. ASCT consisted of 2.5×10⁸ nucleated bonemarrow cells/kg, infused intravenously (IV) on day 0.

On day 1 following ASCT (day +1), the patient received 10⁴ T cells/kg ofperipheral blood lymphocytes (PBL) from an HLA-matched donor. On days+8, +22, +29 and +36 she received PBL from the same donor at anequivalent dose of 10⁵, 10⁵, 10⁶, and 10⁶ T cells/kg, respectively. Onday +47 she received PBL from the same donor at an equivalent dose of10⁷ T cells/kg. The patient showed no evidence of GVHD.

Patient No. 2

This female patient was diagnosed with AML, FAB M5, and was in firstcomplete remission at the time of ASCT. The patient was 42 years old atthe time of ASCT. Prior to ASCT, she received a conditioning regimen ofBusulfan, 4 mg/kg, (days -9 to -6), as well as Cytoxan, 50 mg/kg, days-5 to -2, Cotrimoxazol, 10 mg/kg, days -10 to -2, Allopurinol, 300mg/kg, days -10 to -1 and Ara-C, 25 mg intrathecally.

Prior to ASCT, the autologous bone marrow cells to be infused werepurged by treatment with ASTA-Z. Autologous cells were resuspended at aconcentration of 20×10⁶ cells/ml in Hank's medium containing 4% humanserum albumin. ASTA-Z was then added to a final concentration of 100ug/ml and the cells were incubated in the ASTA-Z at 37° C. for 30 min.After this, the cells were washed twice in Hank's medium, counted andcryopreserved. ASCT consisted of 1×10⁸ nucleated bone marrow cells/kg,infused IV on day 0.

One day +1, the patient received 10⁴ T cells/kg to PBL from anHLA-matched donor. On days +8, +18 and +26 she received PBL from thesame donor at an equivalent dose of 10⁵, 10⁶, and 10⁷ T cells/kg,respectively. One day +80 she received PBL from the same donor at anequivalent dose of 10⁷ T cells/kg, with 3×10⁶ IU of rhIL-2/m² /day for 3days, by subcutaneous injection beginning on day +80. The patient showedno evidence of GVHD.

Patient No. 3

This female patient was diagnosed with AML, FAB M3, and was in firstcomplete remission at the time of ASCT. The patient was 32 years old atthe time of ASCT. Prior to ASCT, she received a conditioning regimen ofBusulfan, 4 mg/kg, (days -9 to -6), as well as Cytoxan, 50 mg/kg, days-5 to -2, Cotrimoxazol, 10 mg/kg, days -10 to -2, Allopurinol, 300mg/kg, days -10 to -1, and Ara-C, 25 mg intrathecally. ASCT consisted ofnon-purged bone marrow cells, and 0.79×10⁸ nucleated cells/kg wereinfused IV on day 0. On day +1, the patient received PBL from anHLA-matched donor at an equivalent dose of 10⁷ T cells/kg. On day +1,she also received 6×10⁶ IU of rhIL-2/m² by subcutaneous injection. NoGVHD was observed.

Patient No. 4

This male patient was diagnosed with AML, FAB M2, and was in firstcomplete remission at the time of ASCT. The patient was 23 years old atthe time of ASCT. Prior to ASCT, he received a conditioning regimen ofBusulfan 4 mg/kg, days -9 to -6, Cytoxan 60 mg/kg, days -3 to -2,Thiotepa 5 mg/kg/day, days -5 to -4, Cotrimoxazol, 10 mg/kg, days -10 to-2, Allopurinol, 300 mg/kg, days -10 to -1 and Ara-C, 25 mgintrathecally. ASCT consisted of non-purged 1.37×10⁸ nucleated bonemarrow cells/kg, infused IV on day 0. On day +1, the patient receivedPBL from an HLA-matched donor at an equivalent dose of 10⁷ T cells/kg.On day +1, he also received 6×10⁶ IU of rhIL-2/m² by subcutaneousinjection. GVHD was suspected (Grade I) in the skin.

Patient No. 5

This male patient was diagnosed with chronic myelogenous leukemia (CML)in chronic phase. The original CML karyotype was positive for thePhiladelphia chromosome (Ph+). The patient was in chronic phase (CP) andwas Ph- at the time of ASCT. The patient was 57 years old at the time ofASCT. Prior to ASCT, he received a conditioning regimen of total bodyirradiation (TBI) 200 cGy/day, days -5 to -3, and Cytoxan 60 mg/kg, days-2 to -1. ASCT consisted of non-purged 0.5×10⁸ nucleated bone marrowcells/kg, infused IV on day 0.

On day +71, as soon as blood counts had stabilized (WBC: 4,200/mm^(3;)Hb: 11.5 gt; platelets: 133,000/mm³), the patient received 3×10⁷ Tcells/kg of PBL from an HLA-/matched brother. No GVHD was observed;hence; the allo-CMI regimen was escalated. On day +10⁷ , the patientreceived 4.6×10⁷ T cells/kg of PBL from the same donor. Starting on day+10⁷ , he also received 6×10⁶ IU of rhIL2/m² /day for 3 days, bysubcutaneous injection. No GVHD was observed. On day +240, after the Ph+karyotype had reappeared, the patient received 4.95×10⁷ cell/kg ofactivated donor lymphocytes ("ADL") from the same donor. Starting on day+240, he also received 6×10⁶ IU of rhIL-2/m² /day for 3 days, bysubcutaneous injection. The ADL were produced by exposing the donor'sPBL to 6,000 IU/ml rhIL2 for four days in culture.

Patient No. 6

This female patient was diagnosed with AML, FAB M2, and was in firstcomplete remission at the time of ASCT. The patient was 50 years old atthe time of ASCT. Prior to ASCT, she received a conditioning regimen ofBusulfan 4 mg/kg, days -9 to -6, Cytoxan 60 mg/kg, days -3 to -2,Thiotepa 5 mg/kg/day, days -5 to -4, Cotrimoxazol, 10 mg/kg, days -10 to-2, Allopurinol, 300 mg/kg/days -10 to -1 and Ara-C, 25 mgintrathecally. ASCT consisted of non-purged 0.64×10⁸ nucleated bonemarrow cells/kg infused IV on day 0. On day +58, as soon as blood countshad stabilized (WBC:4,400/mm³ ; Hb:9.5 g %; platelets:66,000/mm³), thepatient received 5×10⁷ T cells/kg of PBL from an HLA-matched sister. Onday +86 the patient received a second dose of 6.1×10⁷ cells/kg of PBLfrom the HLA-matched sister. Starting on day +86, she also received6×10⁶ IU of rhIL-2/m² /day for 3 days, by subcutaneous injection. NoGVHD was observed.

Patient No. 7

This male patient was diagnosed with CML. The original CML karyotype wasPh+. The patient was in chronic phase (CP) and 50% of his marrow cellswere Ph+ (i.e., the patient was Ph+) at the time of ASCT. The patientwas 47 years old at the time of ASCT. Prior to ASCT, he received aconditioning regimen of TBI 200 cGY/day, days -5 to -3, and Cytoxan60mg/kg, days -2 to -1. ASCT consisted of non-purged 0.98×10⁸ nucleatedbone marrow cells/kg, infused IV on day 0. On day +55, as soon as bloodcounts had stabilized (WBC:6,900/mm³ ;Hb:12.0 g%;platelets:248,000/mm³), the patient received 4×10⁷ T cells/kg of PBLfrom an HLA-matched sister. No GVHD developed. On day +77, the patientreceived 2.8×10⁷ cells/kg of PBL from the HLA-matched sister. Startingon day +77, he also received 6×10⁶ IU of rhIL-2/m² /day for 3 days, bysubcutaneous injection. No GVHD was observed.

Patient No. 8

This male patient was diagnosed with non-Hodgkin's lymphoma (NHL),Burkitt-like, and was in a second partial remission at the time of ASCT.As used herein, the term "partial remission" indicates at least a 50%response (i.e., at least a 50% reduction of lymphoma cells mass) butwith continued evidence of disease. The patient was 36 years old at thetime of ASCT. Prior to ASCT, he received a conditioning regimen ofetoposide, 200 mg/m² /day, days -6 to -3, thiotepa, 40 mg/m² /day, days-5 to -2, Ara-C, 200 mg/m² /day, days -4 to -1, Cytoxan, 60mg/kg/day,day -3, and melphalan, 60 mg/m² /day, days -2 to -1.

ASCT consisted of 0.74×10⁸ /kg viable bone marrow nucleated cells plus2.36×10⁸ /kg viable peripheral blood stem cells. Subcutaneous GM-CSF, 5ug/kg/day, was administered from day +1 to day +18. Prior to ASCT, theautologous cells were purged with Dynal magnetic beads coated withanti-CD19 for elimination of residual lymphoma cells.

On day +90, the patient received 5×10⁷ cells/kg of PBL from anHLA-matched brother. The patient showed no signs of GVHD following thisfirst cell infusion. Polymerase chain reaction (PCR) analysis using twoVNTR loci (VNTR =Variable Number of Tandem Repeats) revealed no evidenceof circulating donor-specific cells. On day +124, the patient received5×10⁷ cells/kg of PBL from the same donor. This was followed by threedays of outpatient treatment with 6×10⁶ IU of rhIL-2/m² /day, bysubcutaneous injection, beginning on day +124.

Fifty days later (day +174) the patient developed pancytopenia, and bonemarrow biopsy revealed severe hypocellular marrow with increased numbersof large granular lymphocytes and plasma cells. Lymphocytes with asimilar morphology were found in the blood. Repeated PCR using twodifferent VNTR loci showed partial engraftment of donor cells on day+191 and 100% engraftment of donor cells on day +211. An allogeneic BMTwas then performed by infusing the patient with 4.2×10⁸ /kg of thedonor's marrow cells; no post-transplant anti-GVHD prophylaxis wasadministered. The patient had rapid three-lineage engraftment withnormal platelet counts after 14 days and normal hemoglobin after 26days. WBC normalized after 10 days with 70% neutrophil, 5% monocytes and25% lymphocytes. Large granular lymphocytes disappeared from the blood.On day 14 following allogeneic BMT, the patient showed minimal signs ofacute GVHD with involvement of skin and oral cavity. There was nointestinal or liver involvement. Since then the patient has continue toexperience grade I/II mucocutaneous GVHD, partially controlled withsteroids and cyclosporin A.

Patient No. 9

This male patient was diagnosed with NHL, follicular mixed IV A, and wasin a second partial remission at the time of ASCT. The patient was 39years old at the time of ASCT. Prior to ASCT, he received a conditioningregimen of etoposide, 200 mg/m² /day, days -6 to -3; thiotepa, 40 mg/m²/day, days -5 to -2; Ara-C, 200 mg/m² /day, days -4 to -1; Cytoxan, 60mg/kg/day, day -3; and melphalan, 60 mg/m² /day, days -2 to -1.

ASCT consisted of 0.5×10⁸ nucleated bone marrow cells/kg, infused IV onday 0. Prior to ASCT, the autologous cells were purged with Dynalmagnetic beads coated with anti-CD19 for elimination of contaminatingtumor cells.

At week 8 post-ASCT, the patient received 5×10⁷ T cells/kg of PBL froman HLA-matched sister. At week 12 post-ASCT, the patient received 5×10⁷T cells/kg of PBL from the same donor. Starting at week 12, on the sameday as the administration of the second Allo-CMI treatment, the patientalso received 6×10⁶ IU of rhIL-2/m² /day for 3 days, by subcutaneousinjection. No GVHD was observed. At week 16 post-ASCT, the patientreceived 0.5×10⁷ ADL/kg from the HLA-matched sister. The ADL wereproduced by exposing the donor's PBL to 6,000 IU/ml rhIL-2 for four daysin culture. No GVHD was observed.

Patient No. 10

This female patient was diagnosed with NHL, mixed cellularity II A, andwas in a second complete remission at the time of ASCT. The patient was36 year old at the time of ASCT. Prior to ASCT, she received aconditioning regimen of etoposide, 200 mg/m² /day, days -6 to -3,thiotepa, 40 mg/m² /day, days -5 to -2, Ara-C, 200 mg/m² /day, days -4to -1, Cytoxan, 60 mg/kg/day, day -3, and melphalan, 60 mg/m² /day, days-2 to -1.

ASCT consisted of 0.94×10⁸ non-purged nucleated bone marrow cells/kgplus 3.9×10⁷ peripheral blood stem cells mobilized by G-CSF prior tocollection with the CS 3000® Plus blood cell separator (BaxterHealthcare Corporation, Fenwal System Catalogue No. 4R4538). Cells wereinfused IV on day 0.

At week 10 post-ASCT, as soon as blood counts had stabilized(WBC:4,300/mm³ ; Hb: 11.2 g %; platelets: 116,000/mm³), the patientreceived 3×10⁷ T cells/kg of PBL from an HLA-matched brother. At week 15post-ASCT, the patient received 4.1×10⁷ T cells/kg of PBL from the samedonor. Starting at week 15, on the same day as the administration of thesecond Allo-CMI treatment, the patient also received 6×10⁶ IU ofrhIL-2/m² /day for 3 days, by subcutaneous injection. At week 23post-ASCT, the patient received 5×10⁷ ADL/kg from the HLA-matchedbrother. The ADL were produced by exposing the donor's PBL to 6,000IU/ml rhIL-2 for four days in culture. Starting at week 23, on the sameday as the administration of the Allo-CCI treatment, the patient alsoreceived 6×10⁶ IU of rhIL-2/m² /day for 3 days, by subcutaneousinjection. No GVHD was observed.

Patient No. 11

This female patient was diagnosed with NHL, immunoblastic, and was in asecond complete remission at the time of ASCT. The patient was 21 yearsold at the time of ASCT. Prior to ASCT, she received a conditioningregimen of etoposide, 200 mg/m² /day, days -6 to -3; thiotepa, 40 mg/m²/day, days -5 to -2; Ara-C, 200 mg/m² /day, days -4 to -1; Cytoxan, 60mg/kg/day, day -3; and melphalan, 60 mg/m² /day, days -2 to -1.

ASCT consisted of 3.82×10⁸ non-purged bone marrow cells/kg plus 1.29×10⁸peripheral blood stem cells mobilized with G-CSF prior to collectionwith the CS 3000® Plus blood cell separator. Cells were infused IV onday 0.

At week 10 post-ASCT, the patient received 5×10⁷ T cells/kg of PBL froman HLA-matched sister. At week 15 post-ASCT, the patient received asecond infusion of 5×10⁷ T cell/kg of PBL from the HLA-matched sister.At week 19 post-ASCT, the patient received a third infusion of 5×10⁷ Tcells/kg of PBL from the same donor. No GVHD was observed, but thepatient did not accept the suggestion that she receive in vivo rhIL2. Atweek 30 post-ASCT, the patient received a fourth infusion of 5×10⁷ Tcells/kg of PBL from the HLA-matched sister. Starting at week 23, on thesame day as the administration of the fourth Allo-CMI treatment, thepatient also received 6×10⁶ IU of rhIL-2/m² /day for 3 days, bysubcutaneous injection. No GVHD has been observed to date.

Patient No. 12

This female patient was diagnosed with NHL, diffuse large cell, and wasin condition of relapse at the time of ASCT. The patient was 21 yearsold at the time of ASCT. Prior to ASCT, she received a conditioningregimen of etoposide, 200 mg/m² /day, days -6 to -3; thiotepa, 40 mg/m²/day, days -5 to -2; Ara-C, 200 mg/m² /day, days -4 to -1; Cytoxan, 60mg/kg/day, day -3; and melphalan, 60 mg/m² /day, days -2 to -1.

ASCT consisted of 1.8×10⁸ non-purged mononuclear bone marrow cells/kg,infused IV on day 0.

At week 6 post-ASCT, as soon as blood counts had stabilized (WBC:4,400/mm^(3;) Hb:11.7 g %; platelets: 150,000/mm³), the patient received5×10⁷ T cells/kg of PBL from a single locus-mismatched sister. At week10 post-ASCT, the patient received a second infusion of 5×10⁷ T cell/kgof PBL from the same donor. Starting at week 10, on the same day as theadministration of the second Allo-CMI treatment, the patient alsoreceived 6×10⁶ IU of rhIL-2/m^(2/) day for 3 days, by subcutaneousinjection. No GVHD was observed.

Patient No. 13

This male patient was diagnosed with AML, FAB M5, and was in firstcomplete remission at the time of ASCT. The patient was 46 years old atthe time of ASCT. Prior to ASCT, he received a conditioning regimen ofBusulfan 4/ mg/kg, days -9 to -6; Cytoxan 60 mg/kg, days -3 to -2;Thiotepa, 5 mg/kg/day, days -5 to -4; Cotrimoxazol, 10 kg/mg, days -10to -2, Allopurinol, 300 mg/kg, days -10 to -1 and Ara-C, 25 mgintrathecally. ASCT consisted of 1.39×10⁸ non-purged bone marrow cellsinfused IV on day 0. At week 11, the patient received 3.86×10⁷ Tcells/kg of PBL from an HLA-matched brother. No GVHD developed.

Patient No. 14

This female patient was diagnosed with AML, FAB M3, and was in secondpartial remission at the time of ASCT. The patient was 12 years old atthe time of ASCT. Prior to ASCT, she received a conditioning regimen ofBusulfan 4 mg/kg, days -9 to -6; Cytoxan 60 mgkg, days -3 to -2;Mitoxantrone 6 mg/m² /day, days -5 to -4; Cotrimoxazol, 10 mg/kg, days-10 to -2, Allopurinol, 300 mg/kg, days -10 to -1 and Ara-C, 25 mgintrathecally. ASCT consisted of 1.14×10⁸ non-purged marrow cellsinfused IV on day 0. On day +113, the patient received 2×10⁷ T cells/kgof single locus-mismatched PBL from her mother. On day +142, the patientreceived 1000 mg/m² Cytoxan in order to improve the efficacy andtemporal engraftment of a second infusion of the donor's PBL. Twentyfour hours later, the patient received 1.7×10⁷ T cells/kg from the samedonor. Starting on the same day as the administration of the secondAllo-CMI treatment, the patient also received 6×10⁶ IU of rhIL-2/m² /dayfor 3 days, by subcutaneous injection. On day +188, the patient received1.5×10⁷ ADL/kg from the same donor. The ADL were produced by exposingthe donor's PBL to 6,000 IU/ml rhIL-2 for four days in culture. On thesame day as the administration of the Allo-CCI treatment, the patientalso received 6×10⁶ IU of rhIL-2/m² /day for 3 days, by subcutaneousinjection.

Patient No. 15

This male patient was diagnosed with AML, FAB M5, and was in firstcomplete remission at the time of ASCT. The patient was 6 1/2 years oldat the time of ASCT. Prior to ASCT, he received a conditioning regimenof Busulfan 4 mg/kg, days -9 to -6; Cytoxan 60 mg/kg, days -3 to -2;Thiotepa 5 mg/kg/day, days -5 to -4; Cotrimoxazol, 10 mg/kg, days -10 to-2, Allopurinol, 300 mg/kg, days -10 to -1 and Ara-C, 25 mgintrathecally. ASCT consisted of 2.7×10⁸ non-purged marrow cells infusedIV on day 0. At week 14, the patient received 5×10⁷ T cells/kg of singlelocus-mismatched PBL from his father. The patient later (May 3, 1994)received 2.5×10⁷ T cells/kg of single locus-mismatched PBL from the samedonor. On the same day as the administration of the second Allo-CMItreatment, the patient also received 6×10⁶ IU of rhIL-2/m² /day for 3days, by subcutaneous injection. Prior to the second Allo-CMI treatment,the patient received cytoxan at 500-1,000 mg/m² with adequate hydration.One month later the patient received 7.8×10⁷ ADL/kg from the same donor.The ADL were produced by exposing the donor's PBL to 6,000 IU/ml rhIL-2for four days in culture. Beginning on the same day as theadministration of the Allo-CCI treatment, the patient also received6×10⁶ IU of rhIL-2/m² /day for 3 days, by subcutaneous injection. Priorto the Allo-CCI treatment, the patient received cytoxan at 500-1,000mg/m² with adequate hydration.

Patient No. 16

This male patient was diagnosed with NHL, mixed large and small cell, inthe lymph nodes, in February of 1993. There was also heavy bone marrowinvolvement. He received chemotherapy (12 courses of MACOP-B), butrelapsed in the lymph nodes and bone marrow in October of 1993. Heunderwent additional chemotherapy and was in a second partial remissionat the time of ASCT, with recurrence in the marrow. The patient was 45years old at the time of ASCT. Prior to ASCT, he received a conditioningregimen consisting of etoposide, 200 mg/m² /day, days -6 to -3;thiotepa, 40 mg/m² /day, days -5 to -2; Ara-C, 200 mg/m² /day, days -4to -1; Cytoxan, 60 mg/kg/day, day -3; and melphalan, 60 mg/m² /day, days-2 to -1.

ASCT consisted of 2.15×10⁸ non-purged G-CSF-mobilized peripheral bloodstem cells, infused IV on day 0.

At week 7 post-ASCT, the patient received 3×10⁷ T cells/kg of PBL froman HLA-matched sister. At week 11 post-ASCT, the patient received asecond infusion of 3×10⁷ T cell/kg of PBL from the same donor. Startingat week 11, on the same day as the administration of the second Allo-CMItreatment, the patient also received 6×10⁶ IU of rhIL-2/m² /day for 3days, by subcutaneous injection. Prior to the second Allo-CMI treatment,the patient received cyclophosphamide (cytoxan) at 500-1,000 mg/m² withadequate hydration. No GVHD was observed. To escalate the treatment, thepatient received 3×10⁷ ADL/kg from the HLA-matched donor. The ADL wereproduced by exposing the donor's PBL to 6,000 IU/ml rhIL-2 for four daysin culture. On the same day as the administration of the Allo-CCItreatment, the patient also received 6×10⁶ IU of rhIL-2/m² /day for 3days, by subcutaneous injection. Prior to the Allo-CCI treatment, thepatient received melphalan at 30 mg/m².

Notes on Patient No.'s 17-23

Patients 17-23 represent additional lymphoma patients treated similarlyto patients 8-12 and 16, except that the initial infusion of allogeneiclymphocytes was accompanied by in vivo administration of rhIL2. Relevantaspects of the treatment protocols are summarized below:

Patient No. 17

This male patient was diagnosed with Hodgkins Disease, with nodularsclerosis, and was in a third partial remission at the time of ASCT. Hewas 16 years old at the time of ASCT. Prior to ASCT, he received aconditioning regimen as set out above for patient No. 16. The ASCTconsisted of bone marrow cells only. Following partial hematopoieticrecovery, the patient received 3×10⁷ T cells/kg of PBL from anHLA-compatible sibling. On the same day as the administration of thefirst Allo-CMI treatment, the patient also received 6×10⁶ IU ofrhIL-2/m² /day for 3 days, by subcutaneous injection. Prior to theAllo-CMI treatment, the patient received cytoxan at 500-1,000 mg/m² withadequate hydration. No GVHD was observed. To escalate the treatment, thepatient received 2.0×10⁷ ADL/kg from the HLA-matched donor. The ADL wereproduced by exposing the donor's PBL to 6,000 IU/ml rhIL-2 for four daysin culture. On the same day as the administration of the Allo-CCItreatment, the patient also received 6×10⁶ IU of rhIL-2/m² /day for 3days, by subcutaneous injection. Prior to the Allo-CCI treatment, thepatient received cytoxan at 500-1,000 mg/m² with adequate hydration.

Patient No. 18

This female patient was diagnosed with NHL, Low Grade, and was in secondcomplete remission at the time of ASCT. She was 24 years old at the timeof ASCT. Prior to ASCT, she received a conditioning regimen as set outabove for patient No. 16. The ASCT consisted of bone marrow cells(1.95×10⁸ /kg) plus peripheral blood stem cells (3.88×10⁸ /kg). On day+169, the patient received 3.6×10⁷ T cells/kg of PBL from anHLA-compatible sister. On the same day as the administration of thefirst Allo-CMI treatment, the patient also received 6×10⁶ IU ofrhIL-2/m² /day for 3 days, by subcutaneous injection. Prior to theAllo-CMI treatment, the patient received cytoxan at 750 mg/m² withadequate hydration. No GVHD was observed. To escalate the treatment, thepatient received (day +171) 3.0×10⁷ ADL/kg from the HLA-matched donor.The ADL were produced by exposing the donor's PBL to 6,000 IU/ml rhIL-2for four days in culture. On the same day as the administration of theAllo-CCI treatment, the patient also received 6×10⁶ IU of rhIL-2/m² /dayfor 3 days, by subcutaneous injection. Prior to the Allo-CCI treatment,the patient received melphalan at 30 mg/m².

Twelve (12) days after administration of the ADL, the patient developedsevere bone marrow aplasia. An allogeneic bone marrow transplant wasperformed with the matched PBL donor's bone marrow cells (2.9×10⁸ /kg),depleted of immunocompetent T cells using the Campath-1G, a monoclonalrat anti-human CDW52 (IgG2b) antibody, provided by Drs. G. Hale and H.Waldmann (Oxford University, UK). See, e.g., Hale et al., Campath-1monoclonal antibodies in bone marrow transplantation. J. Hematotherapy3: 15-31 (1994). The Campath-1G antibody was used at a concentration of0.3 μg/10⁶ nucleated cells. Granulocyte reconstitution was enhanced bydaily subcutaneous administration of G-CSF (5 ug/kg/day). Subsequent tothe allogeneic BMT, the patient totally recovered with normal 3-lineageengraftment of 100% donor-type hematopoietic cells and no residual hostDNA, as attested to by VNTR-PCR. There was no acute GVHD, and noanti-GVHD prophylaxis was used.

Patient No. 19

This female patient was diagnosed with NHL, High Grade, with refractorydisease. She was 48 years old at the time of ASCT. Prior to ASCT, shereceived a conditioning regimen as set out above for patient No. 16. TheASCT consisted of bone marrow cells and peripheral blood stem cellscombined. Following partial hematopoietic recovery, the patient received5.8×10⁷ T cells/kg of PBL from an HLA-compatible sibling. On the sameday as the administration of the first Allo-CMI treatment, the patientalso received 6×10⁶ IU of rhIL-2/m² /day for 3 days, by subcutaneousinjection. No GVHD was observed. To escalate the treatment, the patientreceived 3.8×10⁷ ADL/kg from the HLA-matched donor. The ADL wereproduced by exposing the donor's PBL to 6,000 IU/ml rhIL-2 for four daysin culture. On the same day as the administration of the Allo-CCItreatment, the patient also received 6×10⁶ IU of rhIL-2/m² /day for 3days, by subcutaneous injection. Prior to the Allo-CCI treatment, thepatient received melphalan at 30 mg/m².

Patient No. 20

This male patient was diagnosed with Hodgkins Disease, with nodularsclerosis, and was in a second complete remission at the time of ASCT.He was 49 years old at the time of ASCT. Prior to ASCT, he received aconditioning regimen as set out above for patient No. 16. The ASCTconsisted of bone marrow cells only (0.9×10⁸ /kg). On day +183, thepatient received 8.3×10⁶ T cells/kg of PBL from an HLA-compatiblesister. On the same day as the administration of the Allo-CMI treatment,the patient also received 6×10⁶ IU of rhIL-2/m² /day for 3 days, bysubcutaneous injection. Prior to the Allo-CMI treatment, the patientreceived cytoxan at 1,500 mg/m² with adequate hydration.

Twenty one (21) days following the Allo-CMI treatment, the patientdeveloped bone marrow aplasia. An allogeneic bone marrow transplant wasperformed with the matched PBL donor's bone marrow cells (3.3×10⁸ /kg),depleted of immunocompetent T cells using Campath-1G (0.3 μg/10⁶nucleated cells).

Patient No. 21

This female patient was diagnosed with NHL, High Grade, and was insecond partial remission at the time of ASCT. She was 39 years old atthe time of ASCT. Prior to ASCT, she received a conditioning regimen asset out above for patient No. 16. The ASCT consisted of bone marrowcells and peripheral blood stem cells combined. Prior to ASCT, theautologous cells were purged with Dynal magnetic beads coated withanti-CD19 for elimination of contaminating tumor cells. Followingpartial hematopoietic recovery, the patient received 2.7×10⁷ T cells/kgof PBL from an HLA-compatible sibling. On the same day as theadministration of the first Allo-CMI treatment, the patient alsoreceived 6×10⁶ IU of rhIL-2/m² /day for 3 days, by subcutaneousinjection. Prior to the Allo-CMI treatment, the patient received cytoxanat 500-1,000 mg/m² with adequate hydration.

Patient No. 22

This male patient was diagnosed with Hodgkins Disease, with nodularsclerosis, and was in a third complete remission at the time of ASCT. Hewas 29 years old at the time of ASCT. Prior to ASCT, he received aconditioning regimen as set out above for patient No. 16. The ASCTconsisted of bone marrow cells only (0.64×10⁸ /kg). On day +110, thepatient received 4×10⁷ T cells/kg of PBL from an HLA-compatible sister.On the same day as the administration of the Allo-CMI treatment, thepatient also received 6×10⁶ IU of rhIL-2/m² /day for 3 days, bysubcutaneous injection. Prior to the Allo-CMI treatment, the patientreceived melphalan at 15 mg/m² with adequate hydration. On day +150, thepatient received 3.3×10⁷ ADL/kg from the HLA-matched donor. The ADL wereproduced by exposing the donor's PBL to 6,000 IU/ml rhIL-2 for four daysin culture. On the same day as the administration of the Allo-CCItreatment, the patient also received 6×10⁶ IU of rhIL-2/m² /day for 3days, by subcutaneous injection. Prior to the Allo-CCI treatment, thepatient received melphalan at 30 mg/m².

Six (6) days following the Allo-CCI treatment, the patient developedbone marrow aplasia. An allogeneic bone marrow transplant was performedwith the matched PBL donor's bone marrow cells (3.52×10⁸ /kg), depletedof immunocompetent T cells using Campath-1G (0.1 μg/10⁶ nucleatedcells).

Patient No. 23

This male patient was diagnosed with NHL, Intermediate Grade, and was infirst partial remission at the time of ASCT. He was 38 years old at thetime of ASCT. Prior to ASCT, he received a conditioning regimen as setout above for patient No. 16. The ASCT consisted of bone marrow cellsand peripheral blood stem cells combined. Following partialhematopoietic recovery, the patient received 2.8×10⁷ T cells/kg of PBLfrom an HLA-compatible sibling. On the same day as the administration ofthe first Allo-CMI treatment, the patient also received 6×10⁶ IU ofrhIL-2/m² /day for 3 days, by subcutaneous injection.

Notes on Patient No.'s 24-29

Patients 24-29 are breast cancer patients who were treated with adebulking regimen including ASCT. This was followed by Allo-CMI and, insome cases, Allo-CCI. Six patients with metastatic disease, with verygrave prognoses, were offered immunotherapy according to the presentinvention. The advanced nature of the malignancies tempered expectationsof complete disease eradication, but in the absence of viabletherapeutic alternatives, the decision was made to proceed. Of the sixpatients, four were with evidence of major metastatic disease at thetime immunotherapy was initiated. None of these patients showed evidenceof engraftment. As such, the infused allogeneic cells may not have hadsufficient time to mount a full GVT response. Even in mice, a residencetime for the infused allogeneic cells of 2-3 weeks is required to obtaintumor elimination. Weiss et al., Effective Graft vs. Leukemia EffectsIndependently of Graft vs. Host Disease Following T-Cell depletedallogeneic bone marrow transplantation in a murine model of B-CellLeukemia/Lymphoma (BCL1): Role of Cell Therapy and rHIL-2. J. Immunology153(6): 2562-7 (Sept. 1994). Nevertheless, as described below, one ofthese patients showed a pronounced, though transient, response to celltherapy.

Two of the metastatic breast cancer patients entered the study inremission, after elimination of tumor bulk by chemotherapy prior to celltherapy. As described below, both of these patients show no evidence ofdisease over 11 and over 9 months following ASCT, are free of anysymptoms, and have Karnofsky scores of 100%. This is in spite of thefact that, again, there was no evidence of engraftment. With futurepatients, engraftment will be possible with more aggressive applicationof the cell therapy.

Patient No. 24

This female patient was diagnosed with metastatic breast cancer, withmetastases to the liver and spine. She underwent a right partialmastectomy with axillary lymph node dissection showing involvement of18/35 lymph nodes. The patient was 43 years old at the time of ASCT.Prior to ASCT, she received a conditioning regimen of carboplatin, 200mg/m² /day, days -7 to -4; thiotepa, 60 mg/m² /day, days -6 to -4;etoposide, 200 mg/m² /day, days -5 to -3; and melphalan, 60 mg/m² /day,days -4 to -3.

At the time of ASCT, the patient showed elevation of the breast cancercell marker CA-15.3. ASCT consisted of 3.74×10⁸ peripheral blood stemcells/kg, infused IV on day 0. The stem cells were collected aftermobilization with G-CSF (7.5 mg/kg/day for 5 days) using threecollections of the CS 3000® Plus blood cell separator. Following ASCT,the patient recovered with no complications and was discharged on day 20post-ASCT.

A fully HLA-matched (A, B, DR and mixed lymphocyte reaction(MLR)-negative) sibling was chosen as donor of PBL for Allo-CMI. On day+77, when the patient had achieved a stable clinical condition (WBC:2,700/mm^(3;) Hb: 9.1 g %; platelets: 56,000/mm³), the patient received3.2×10⁷ T cells/kg of PBL from an HLA-matched sibling. Starting on thesame day as the administration of the first Allo-CMI treatment, thepatient also received 6×10⁶ IU of rhIL-2/m² /day for 3 days, bysubcutaneous injection. On day +117, the patient received 5.4×10⁷ Tcells/kg of ADL, together with in vivo rhIL-2 as described above. ADLwere produced as described above. On day +162, the patient received1.11×10⁸ T cells/kg of PBL (not ADL), together with in vivo rhIL-2 asdescribed above. On day +165, the patient received 8.6×10⁷ T cells/kg ofADL, together with in vivo rhIL-2 as described above. ADL were producedas described above. On day +343, the patient received 1.72×+10⁸ Tcells/kg of ADL (expanded in vitro with phytohaemagglutinin), togetherwith in vivo rhIL-2 as described above.

Patient No. 25

This male patient was diagnosed with metastatic breast cancer, withmetastases to the bone marrow, sternum and vertebrae T₄ to T₇. Heunderwent lumpectomy with axillary lymph node dissection showinginvolvement of 16/18 lymph nodes. The patient was 36 years old at thetime of ASCT. Prior to ASCT, he received a conditioning regimen ofcarboplatin, 200 mg/m² /day, days -7 to -4; thiotepa, 60 mg/m² /day,days -6 to -4; etoposide, 200 mg/m² /day, days -5 to -3; and melphalan,60 mg/m² /day, days -4 to -3.

ASCT consisted of 1.64×10⁸ peripheral blood stem cells/kg, infused IV onday 0. The stem cells were collected after mobilization with G-CSF (7.5mg/kg/day for 5 days) using three collections of the CS 3000® Plus bloodcell separator. Following ASCT, the patient recovered with nocomplications and was discharged on day 20 post-ASCT.

A fully HLA-matched (A, B, DR and mixed lymphocyte reaction(MLR)-negative) brother was chose as donor of PBL for Allo-CMI. On day+33, as soon as the patient had achieved a stable clinical condition(WBC: 11,000/mm^(3;) Hb: 11.5 gt; platelets: 201,000/mm³), the patientreceived 2.3×10⁷ T cells/kg of PBL from an HLA-matched brother. Startingon the same day as the administration of the first Allo-CMI treatment,the patient also received 6×10⁶ IU of rhIL-2/m² /day for 3 days, bysubcutaneous injection. On day +90, the patient received 9.4×10⁶ ADL/kgfrom the same donor. The ADL were produced by exposing the donor's PBLto 6,000 IU/ml rhIL-2 for four days in culture. On the same day as theadministration of the Allo-CCI treatment, the patient also received6×10⁶ IU of rhIL-2/m² /day for 3 days, by subcutaneous injection. On day+170, the patient received 6.7×10⁷ T cells/kg of PBL from the samedonor. On the same day as the administration of the Allo-CCI treatment,the patient also received 6×10⁶ IU of rhIL-2/m² /day for 3 days, bysubcutaneous injection.

Patient No. 26

This female patient was diagnosed with metastatic breast cancer, withinvolvement of 18/30 lymph nodes. The patient was 36 years old at thetime of ASCT, with no evidence of disease. Prior to ASCT, she received aconditioning regimen of carboplatin, 200 mg/m² /day, days -7 to -4;thiotepa, 60 mg/m² /day, days -6 to -4; etoposide, 200 mg/m² /day, days-5 to -3; and melphalan, 60 mg/m² /day, days -4 to -3. ASCT consisted of1.86×10⁸ peripheral blood stem cells/kg, infused IV on day 0. Thepatient relapsed five (5) months after ASCT, with bone metastases.

On day +180, the patient received 4×10⁷ T cells/kg of PBL from anHLA-compatible donor. Starting on the same day as the administration ofthe first Allo-CMI treatment, the patient also received 6×10⁶ IU ofrhIL-2/m² /day for 3 days, by subcutaneous injection. On day +210, thepatient received 1.11×10⁸ T cells/kg of PBL from the same donor. On thesame day as the administration of the second Allo-CMI treatment, thepatient also received 6×10⁶ IU of rhIL-2/m² /day for 3 days, bysubcutaneous injection. On day +216, the patient received 3.3×10⁷ Tcells/kg of ADL from the same donor, together with in vivo rhIL-2 asdescribed above. The ADL were produced by exposing the donor's PBL to6,000 IU/ml rhIL-2 for four days in culture.

Patient No. 27

This female patient was diagnosed with metastatic breast cancer, withinvolvement of 11/31 lymph nodes and metastases to the vertebrae. Thepatient was 44 years old at the time of ASCT, with elevation of a breastcancer cell marker, CA-15.3. Prior to ASCT, she received a conditioningregimen of carboplatin, 200 mg/m² /day, days -7 to -4; thiotepa, 60mg/m² /day, days -6 to -4; etoposide, 200 mg/m² /day, days -5 to -3; andmelphalan, 60 mg/m² /day, days -4 to -3. ASCT consisted of 2.79×10⁸peripheral blood stem cells/kg, infused IV on day 0.

On day +210, the patient received 2.98×10⁷ T cells/kg of PBL from anHLA-compatible donor. Starting on the same day as the administration ofthe first Allo-CMI treatment, the patient also received 6×10⁶ IU ofrhIL-2/m² /day for 3 days, by subcutaneous injection. On day +270, thepatient received 6.54×10⁷ ADL/kg from the same donor. The ADL wereproduced by exposing the donor's PBL to 6,000 IU/ml rhIL-2 for four daysin culture. On the same day as the administration of the Allo-CCItreatment, the patient also received 6×10⁶ IU of rhIL-2/m² /day for 3days, by subcutaneous injection.

Patient No. 28

This female patient was diagnosed with metastatic breast cancer, withmetastases to the ribs and vertebrae. The patient was 54 years old atthe time of ASCT. Prior to ASCT, she received a conditioning regimen ofcarboplatin, 200 mg/m² /day, days -7 to -4; thiotepa, 60 mg/m² /day,days -6 to -4; etoposide, 200 mg/m² /day, days -5 to -3; and melphalan,60 mg/m² /day, days -4 to -3. ASCT consisted of 2.69×10⁸ peripheralblood stem cells/kg, infused IV on day 0. The patient showed no evidenceof disease at the time of initiation of Allo-CMI treatment.

On day +125, the patient received 3.3×10⁷ T cells/kg of PBL from anHLA-compatible donor. Starting on the same day as the administration ofthe first Allo-CMI treatment, the patient also received 6×10⁶ IU ofrhIL-2/m² /day for 3 days, by subcutaneous injection.

Patient No. 29

This female patient was diagnosed with metastatic breast cancer, withbone metastases as well as metastases to cervical and supraclavicularlymph nodes. The patient was 51 years old at the time of ASCT. Prior toASCT, she received a conditioning regimen of carboplatin, 200 mg/m²/day, days -7 to -4; thiotepa, 60 mg/m² /day, days -6.to -4; etoposide,200 mg/m² /day, days -5 to -3; and melphalan, 60 mg/m² /day, days -4 to-3. ASCT consisted of 1.71×10⁸ peripheral blood stem cells/kg, infusedIV on day 0. The patient showed no evidence of disease at the time ofinitiation of Allo-CMI treatment.

On day +160, the patient received 3.45×10⁷ T cells/kg of PBL from anHLA-compatible donor. Starting on the same day as the administration ofthe first Allo-CMI treatment, the patient also received 6×10⁶ IU ofrhIL-2/m² /day for 3 days, by subcutaneous injection. On day +211, thepatient received 5×10⁷ ADL/kg from the same donor. The ADL were producedby exposing the donor's PBL to 6,000 IU/ml rhIL-2 for four days inculture. On the same day as the administration of the Allo-CCItreatment, the patient also received 6×10⁶ IU of rhIL-2/m² /day for 3days, by subcutaneous injection.

Portions of the above-described patient treatment protocols aresummarized in Table 1, below. Patients are grouped according to disease(AML, CML,NHL and breast cancer).

                                      TABLE 1.sup.a                               __________________________________________________________________________          Status                                                                  Patient No.                                                                         Pre-ASCT                                                                            Cond. Regimen                                                                        Type ASCT  Allo Cells (T Cells/Kg)                         __________________________________________________________________________    AML:                                                                           1    1CR   BU/CY  ABMT: Purged                                                                             PBL 10.sup.4 - 10.sup.7                                                       (day +1 to +47)                                  2    1CR   BU/CY  ABMT: Purged                                                                             PBL 10.sup.4 - 10.sup.7                                                       (day +1 to +80)                                  3    1CR   BU/CY  ABMT: Non-Purged                                                                         PBL 10.sup.7 + IL2                                                            (day +1)                                         4    1CR   BU/CY + TT                                                                           ABMT: Non-Purged                                                                         PBL 10.sup.7 + IL2                                                            (day +1)                                         6    1CR   BU/CY + TT                                                                           ABMT: Non-Purged                                                                         1. PBL                                                                        2. PBL + IL2                                    13    1CR   BU/CY + TT                                                                           ABMT: Non-Purged                                                                         1. PBL                                          14    2PR   BU/CY + MX                                                                           ABMT: Non-Purged                                                                         1. PBL                                                                        2. Cytoxan/PBL + IL2                            15    1CR   BU/CY + TT                                                                           ABMT: Non-Purged                                                                         1. PBL                                                                        2. Cytoxan/PBL + IL2                                                          3. Cytoxan/ADL + IL2                            CML:                                                                           5    C(Ph-)                                                                              CY/TBI ABMT: Non-Purged                                                                         1. PBL                                                                        2. PBL + IL2                                                                  3. ADL + IL2                                     7    C(Ph+)                                                                              CY/TBI ABMT: Non-Purged                                                                         1. PBL                                                                        2. PBL + IL2                                    NHL:                                                                           8    2PR   ETACM  AMBT + PBSC: Purged                                                                      1. PBL                                                                        2. PBL + IL2                                                                  3. Allogeneic BMT (due                                                        to marrow aplasia)                               9    2PR   ETACM  ABMT: Purged                                                                             1. PBL                                                                        2. PBL + IL2                                                                  3. ADL + IL2                                    10    2CR   ETACM  ABMT + PBSC: Purged                                                                      1. PBL                                                                        2. PBL + IL2                                                                  3. ADL + IL2                                    11    2CR   ETACM  ABMT + PBSC: Purged                                                                      1. PBL                                                                        2. PBL                                                                        3. PBL                                                                        4. PBL + IL2                                    12    Relapse                                                                             ETACM  ABMT: Non-Purged                                                                         1. PBL                                                                        2. PBL + IL2                                    16    2PR   ETACM  ABMT + PBSC:                                                                             1. PBL                                                             Non-Purged 2. Cytoxan/PBL + IL2                                                          3. Melphalan/ADL + IL2                          17    3PR   ETACM  ABMT: Non-Purged                                                                         1. Cytoxan/PBL + IL2                                                          2. Cytoxan/ADL + IL2                            18    2CR   ETACM  ABMT + PBSC:                                                                             1. Cytoxan/PBL + IL2                                               Non-Purged 2. Melphalan/ADL + IL2                                                        3. Allogeneic BMT (due                                                        to marrow aplasia)                              19    Refractory                                                                          ETACM  ABMT + PBSC:                                                                             1. PBL + IL2                                                       Non-Purged 2. Melphalan/ADL + IL2                          20    2CR   ETACM  ABMT: Non-Purged                                                                         1. Cytoxan/PBL + IL2                                                          2. Allogeneic BMT (due                                                        to marrow aplasia)                              21    2PR   ETACM  ABMT + PBSC:                                                                             1. Cytoxan/PBL + IL2                                               Purged                                                     22    3CR   ETACM  ABMT: Non-Purged                                                                         1. Melphalan/PBL + IL2                                                        2. Melphalan/ADL + IL2                                                        3. Allogeneic BMT (due                                                        to marrow aplasia)                              23    1PR   ETACM  ABMT + PBSC:                                                                             1. PBL + IL2                                                       Non-Purged                                                 BREAST CANCER:                                                                24    Metastatic                                                                          CTEM   PBSC: Non-Purged                                                                         1. PBL + IL2                                                                  2. ADL + IL2                                                                  3. ADL + IL2                                                                  4. ADL + IL2                                    25    Metastatic                                                                          CTEM   PBSC: Non-Purged                                                                         1. PBL + IL2                                                                  2. ADL + IL2                                                                  3. ADL + IL2                                    26    Metastatic                                                                          CTEM   PBSC: Non-Purged                                                                         1. PBL + IL2                                                                  2. PBL + IL2                                                                  3. ADL + IL2                                    27    Metastatic                                                                          CTEM   PBSC: Non-Purged                                                                         1. PBL + IL2                                                                  2. ADL + IL2                                    28    Metastatic                                                                          CTEM   PBSC: Non-Purged                                                                         1. PBL + IL2                                          (Remission)                                                             29    Metastatic                                                                          CTEM   PBSC: Non-Purged                                                                         1. PBL + IL2                                          (Remission)             2. ADL + IL2                                    __________________________________________________________________________     .sup.a Terms used in the Table are defined as follows:                        1CR, 2CR: First or second complete remission, respectively.                   1PR, 2PR, 3PR: First, second or third partial remission, respectively.        CP(Ph-): Chronic phase, no cytogenetic evidence of Philadelphia               chromosome.                                                                   CP9Ph+): Chronic phase, cytogenetic evidence of presence of Philadelphia      chromosome.                                                                   BU/CY: Conditioning regimen of Busulfan, 4 mg/kg, days 6 through 9 preASC     (days -9 to -6), as well as Cytoxan (cyclophosphamide), 50 mg/kg, days -5     to -2, Cotrimoxazol, 10 mg/kg, days -10 to -2, Allopurinol, 300 mg/kg,        days -10 to -1 and cytosine arabinoside (AraC), 25 mg intrathecally.          BU/CY + TT: Conditioning regimen of Busulfan 4 mg/kg, days -9 to -6,          Cytoxan 60 mg/kg, days -3 to -2, Thiotepa 5 mg/kg/day, days -5 to -4;         Cotrimoxazol, 10 mg/kg, days -10 to -2, Allopurinol, 300 mg/kg, days -10      to -1 and AraC, 25 mg intrathecally.                                          BU/CY + MX: Conditioning regimen of Busulfan 4 mg/kg, days -9 to -6;          Cytoxan 60 mg/kg, days -3 to -2; Mitoxantrone 6 mg/m.sup.2 /day, days -5      to -4; Cotrimoxazol, 10 mg/kg, days -10 to -2, Allopurinol, 300 mg/kg,        days -10 to -1 and AraC, 25 mg intrathecaly.                                  CY/TBI: Conditioning regimen of total body irradiation (TBI) 200 cGY/day,     days -5 to -3, and Cytoxan 60 mg/kg, days -2 to -1.                           ETACM: Conditioning regimen of etoposide, 200 mg/m.sup.2 /day, days -6 to     -3; thiotepa, 40 mg/m.sup.2 /day, days -5 to -2; AraC, 200 mg/m.sup.2         /day, days -4 to -1; Cytoxan, 60 mg/kg/day -3; and melphalan, 60              mg/m.sup.2 /day, days -2 to -1.                                               CTEM: Conditioning regimen of carboplatin, 200 mg/m.sup.2 /day, days -7 t     -4; thiotepa, 60 mg/m.sup.2 /day, days -6 to -4; etoposide, 200 mg/m.sup.     /day, days -5 to -3; and melphalan, 60 mg/m.sup.2 /day, days -4 to -3.        ABMT: Infusion of stem cells extracted directly from the bone marrow.         PBSC: Infusion of stem cells taken from peripheral blood following            mobilization from the bone marrow.                                            PBL, ADL: Without further numerical designation, PBL (peripheral blood        lymphocytes) and ADL (Activated donor lymphocytes) refer to one infusion      of ≧10.sup.7 cells/kg of patient body weight. For patient No.'s        5-18, the first infusion of allogeneic cells was done only after              attainment of partial hematopoiesis reconstitution.                           Cytoxan/PBL or ADL: Cyclophosphamide (Cytoxan) administered prior to          infusion of ≧10.sup.7 cells/kg of PBL or ADL.                          Melphalan/PBL or ADL: Melphalan administered prior to infusion of             ≧10.sup.7 cells/kg of PBL or ADL.                                 

II. RESULTS

The above-described patients have been followed for various lengths oftime. The disease status of the patients is summarized below:

Patient No. 1 relapsed after 6 months and died at 9 months after ASCT.

Patient No. 2 relapsed after 12 months and died at 20 months after ASCT.

Patient No. 3 relapsed after 36 months. After receiving an allogeneicbone marrow transplant, the patient went into a complete remission, butdied of GVHD 6 weeks after the allogeneic BMT.

Patient No. 4 is alive and well 36 months after ASCT, with a Karnofskyscore of 100%.

Patient No. 5 is alive and hematologically in complete remission with nochemotherapy requirement and Karnofsky score of 100%, over 34 monthsafter ASCT. PCR for the bcr/abl translocation characteristic of thePhiladelphia chromosome fluctuates between negative and positive,although the patient's disease appears fully controlled. The patient isreceiving 9×10⁶ units of IFNα (Roferon A, S.C.) every day.

Patient No. 6 is alive and well over 35 months after ASCT with noevidence of disease and Karnofsky score of 100%.

Patient No. 7 is alive and hematologically in complete remission with nochemotherapy requirement and Karnofsky score of 100%, over 33 monthspost-ASCT. Currently, the patient is on IFNα treatment. PCR for thebcr/abl translocation characteristic of the Philadelphia chromosomefluctuates between negative and positive, although the patient's diseaseappears fully controlled.

Patient No. 8 is alive and well over 36 months after initial ASCT andover two years following allogeneic BMT, with no evidence of lymphoma.The patient has mild cutaneous chronic GVHD with Karnofsky score of100%.

Patient No. 9 relapsed after 18 months post-ASCT. The patient is beingtreated with Allo-CMI/CCI from another HLA-matched donor.

Patient No. 10 is alive and well over 29 months after ASCT with noevidence of disease.

Patient No. 11 is alive and well over 28 months after ASCT with noevidence of disease.

Patient No. 12 underwent relapse at 3 months post-ASCT and died at 5months post-ASCT.

Patient No. 13 underwent relapse at 4 months post-ASCT. He did not havea chance to receive allo-CMI with rhIL2 due to severe unrelatedpulmonary complications. The patient died 5 months after relapse.

Patient No. 14 is alive and well with no evidence of disease over 13months post-ASCT, with Karnofsky score of 100%. PCR analysis of bloodshows no evidence of RAR-alpha, the typical molecular marker for AML.

Patient No. 15 is alive and well with no evidence of disease over 15months post-ASCT, with Karnofsky score of 100%.

Patient No. 16 relapsed after 7 months post-ASCT. The patient is aliveand clinically well with evidence of disease at 14 months post-ASCT. Hewas not referred for further treatment and is being treated by hisreferring physician.

Patient No. 17 is alive and well with no evidence of disease over 15months post-ASCT.

Patient No. 18 is alive and well with no evidence of disease over 14months post-ASCT.

Patient No. 19 is alive and well with no evidence of disease over 13months post-ASCT.

Patient No. 20 died of multi-organ failure 13 days following allogeneicBMT for marrow aplasia.

Patient No. 21 is alive and well with no evidence of disease over 9months post-ASCT.

Patient No. 22 died of pneumonia and Candida sepsis 9 days followingallogeneic BMT for marrow aplasia.

Patient No. 23 relapsed at 5 months post-ASCT. He is being treated withradiation therapy to involved areas and will be treated withimmunotherapy as soon as the radiation treatments are over.

Patient No. 24 showed a marked partial remission of 3 months duration,during which time there was a distinctive disappearance of livermetastases from CT scan images (see FIG. 5), and there was a markeddecline in the level of the breast cancer cell marker 15.3 (118 down toless than 4). The patient has relapsed and is now in a state of diseaseprogression.

Patient No. 25 has progressive disease.

Patient No. 26 has progressive disease.

Patient No. 27 has progressive disease.

Patient No. 28 is alive and well with no evidence of disease over 11months post-ASCT.

Patient No. 29 is alive and well with no evidence of disease over 9months post-ASCT.

The results reported above indicate that Allo-CMI and Allo-CCI may bethe most rational and practical approached for eradication of residualmalignant cells. GVT effects induced by administration of allogeneiclymphocytes may be further enhanced by administration in vivo of rhIL2.

EXAMPLE 3 Utility of Cytokines Other Than IL2

Cytokines used in the following experiments were obtained as follows:(1) rhIL2 was provided by Dr. C. R. Franks (EuroCetus BY, Amsterdam, TheNetherlands) as a lyophilized powder in 1 mg vials containing 18×10⁶international units (IU). (2) Recombinant interferon-gamma (rIFNγ) wasprovided by Roussel Uclaf (Romainville, France) as a lyophilized powdercontaining 2×10⁷ U/mg. (3) Recombinant human IL-6 (rIL-6) was kindlyprovided by Dr. M. Revel and Dr. O. Laub (InterPharm Laboratories,Rehovot., Israel) in a concentration of 1.13 mg/ml protein containing43×10⁶ IU/ml. (4) Recombinant human IL-7 (rhIL-7) was provided by PeproTech (New Jersey, USA) as lyophilized powder and was reconstituted to100 mg/ml.

Lymphocytes were preactivated in vitro with rhIL2 or with combinationsof rhIL2 and other cytokines for four days at 37° C. Concentrations ofcytokines used for in vitro incubation of lymphocytes were as follows:(a) rhIL2: 6000 IU/ml; (b) rIL6: 100-1000 U/ml; (c) rIL7: long/ml; and(d) IFNγ: 1000 U/ml. Anti-tumor effects of ADL collected post-culturingwere assayed against natural killer (NK)-sensitive K562 tumor cells andNK-resistant Daudi tumor cells. The in vitro toxicity was evaluated byspecific chromium release following incubation of effector cells withchromium-labeled tumor cells. Results are presented below as lytic unitsper 10⁶ effector cells, determined for 30% lysis of 5×10³ target cells:

    ______________________________________                                                        Cytotoxicity (Lytic                                                           Units/10.sup.6 Cells)                                                         Anti-Daudi                                                                            Anti-K562                                             ______________________________________                                        IL2 alone          48        53                                               IL2 + IL6 + IL7 + IFN                                                                           210       129                                               ______________________________________                                    

EXAMPLE 4 Selected CD8+T Lymphocytes as Agents of the Graft-Versus-TumorEffect

Mice were purchased and maintained as described in EXAMPLE 1. Use andmaintenance of BCL1 leukemia cells were as described in EXAMPLE 1.rhIL-2 was obtained from EuroCetus (Amsterdam, Holland). Concentrationsof rhIL-2 are expressed in International Units (IU), where 6,000 IU isequivalent to 1,000 Cetus Units.

In a first set of experiments, BALB/c x C57BL/6 F1(F1) recipients wereinoculated with 10⁵ BCL1 leukemia cells. BCL1 cells are of BALB/corigin. The recipient mice were conditioned with total body irradiation(TBI) at 750 cGY; 24 hours later the conditioned mice received Allo-CMItreatment using 15×10⁶ spleen cells obtained from C57BL/6 (experimental)or F1 (control) donors. The spleen cells used for Allo-CMI were eitheruntreated or treated with specific monoclonal antibodies to eliminatewell-defined T-cell subsets (CD4 or CD8). CD4+ cells were eliminatedwith the YTS 191 antibody, and CD8+ cells were eliminated with the YTS169 antibody. Both antibodies are IgG2b and, as such, bind effectivelyto the recipient host Fc-positive reticuloendothelial system, resultingin cell lysis through antibody-dependent cell-mediated cytotoxicity(ADCC). The antibodies were provided by Drs. Steve Cobbold and HermanWaldman, Cambridge University, United Kingdom.

In order to assess the effect of Allo-CMI on elimination of tumor cellsin the recipient host, 10⁵ spleen cells from treated recipients at 49days following Allo-CMI were adoptively transferred to secondary BALB/crecipients. Leukemia was followed in secondary recipients in order toassess whether or not spleen cells obtained from the treated animalscontained clonogenic tumor cells.

Results are shown in FIG. 6. The graph is a composite of two experimentsin which 9/10 and 8/10, respectively of recipients treated withCD4-depleted spleen cells remained disease free, whereas 0/7 and 0/10recipients treated with CD8-depleted spleen cells remained disease free.The data indicate that CD8+ cells, but not CD4+ cells, were capable ofeliciting a GVT response in the recipient mice.

In a second set of experiments, T-cell subsets were again evaluated,this time as ADL administered in concert with in vivo administration ofrhIL2. F1 recipients were inoculated with 10⁵ BCL1 leukemia cells.Recipients were conditioned with TBI at 750 cGY. On the following dayeach recipient received 10×10⁶ activated donor lymphocytes (ADL). TheADL (spleen cells activated in vitro with rhIL2 at 6,000 IU/ml for 4days) were given untreated or pretreated with the anti-CD4 and anti-CD8antibodies described above to deplete the cell populations ofwell-defined T-cell subsets. In order to amplify the potentialanti-tumor effects in vivo and to investigate whether or not lack ofwell-defined T-cell subsets may be compensated for by in vivo treatmentwith rhIL-2, recipients were administered 120,000 IU rhIL-2intraperitoneally twice a day for five days.

In order to assess the effect of Allo-CMI on elimination of tumor cellsin the recipient host, 10⁵ spleen cells from treated recipients at 12days following Allo-CMI were adoptively transferred to secondary BALB/crecipients. Leukemia was followed in secondary recipients in order toassess whether or not spleen cells obtained from the treated animalscontained clonogenic tumor cells.

Results are shown in FIG. 7, which summarizes three separateexperiments. The data demonstrate that rhIL-2 can enhance and restorefull GVT activity to ADL even if CD4 cells are depleted. As such, rhIL-2can substitute for CD4 cells under these conditions. In contrast,CD8-depleted ADL cannot be restored to full GVT activity by additionaltreatment i vivo with rhIL-2. These data confirm that the anti-leukemiaeffects are mediated by CD8+ T cells and further indicate that sucheffects can be enhanced by in vivo treatment with rhIL-2.

What is claimed is:
 1. A method of treating a human cancer patienthaving malignant breast cancer cells, said patient having undergone amalignant cell debulking procedure and having further undergoneautologous stem cell transplantation incident to said debulkingprocedure, said patient being at risk for disease relapse due to apopulation of residual malignant breast cancer cells that may remainviable in said patient following said debulking procedure, comprising:a)monitoring said patient until said patient is partially hematopoiesisrecovered but is not fully immune-reconstituted; b) administering tosaid patient allogeneic lymphocytes in a regimen that causes aclinically significant graft-versus-malignant cell response; and c)monitoring said patient for levels of malignant breast cancer cells. 2.The method of claim 1, wherein said lymphocytes are HLA-compatible withsaid patient.
 3. The method of claim 2, wherein said regimen comprisesthe following steps in sequence:a) treating said patient byadministration of about 10⁷ cells/kg to about 10⁹ cells/kg ofHLA-compatible, allogeneic lymphocytes; b) monitoring said patient forindications of a graft-versus-malignant cell response; and c) if no orinsufficient graft-versus-malignant cell response develops in saidpatient, escalating said treatment by performing at least one procedureselected from the group consisting of (1) administration of a number ofHLA-compatible, allogeneic lymphocytes greater than the number oflymphocytes administered in step (a); (2) administration of a number ofHLA-compatible, allogeneic lymphocytes at least as great as the numberof lymphocytes administered in step (a), accompanied by in vivoadministration of at least one T-cell activator to said patient; (3)administration of HLA-compatible, allogeneic ADL to said patient; and(4) administration of HLA-compatible, allogeneic ADL, accompanied by invivo administration of at least one T-cell activator to said patient;wherein more than one of said procedures is performed if no orinsufficient graft-versus-malignant cell response develops in saidpatient following said first or subsequent procedure.
 4. The method ofclaim 3, wherein step (a) of said regimen further comprises in vivoadministration of at least one T-cell activator to said patient.
 5. Themethod of claim 3, wherein said T-cell activator comprises at least oneT-cell signal transduction pathway activator.
 6. The method of claim 5,wherein said T-cell signal transduction pathway activator is selectedfrom the group consisting of IL1, IL2, IL4, IL5, IL6, IL7, IL12, IL13,IFNα, IFNγ, TNFα, anti-CD3, anti-CD28, phytohemagglutinin,concanavalin-A, and phorbol esters.
 7. The method of claim 1 whereinsaid breast cancer cells are metastatic breast cancer cells.
 8. A methodof treating a human cancer patient having malignant breast cancer cells,said patient having undergone a malignant cell debulking procedure andhaving further undergone autologous stem cell transplantation incidentto said debulking procedure, said patient being at risk for diseaserelapse due to a population of residual malignant breast cancer cellsthat may remain viable in said patient following said debulkingprocedure, comprising:a) monitoring said patient until said patient ispartially hematopoiesis recovered but is not fully immune-reconstituted;b) administering to said patient allogeneic lymphocytes in a regimenthat causes a mild graft-versus-host response; and c) monitoring saidpatient for levels of malignant breast cancer cells.
 9. The method ofclaim 8 wherein said breast cancer cells are metastatic breast cancercells.
 10. The method of claim 8, wherein said lymphocytes areHLA-compatible with said patient.
 11. The method of claim 10, whereinsaid regimen comprises the following steps in sequence:a) treating saidpatient by administration of about 10⁷ cells/kg to about 10⁹ cells/kg ofHLA-compatible, allogeneic lymphocytes; b) monitoring said patient forindications of a mild graft-versus-host response; and c) if no orinsufficient graft-versus-host response develops in said patient,escalating said treatment by performing at least one procedure selectedfrom the group consisting of (1) administration of a number ofHLA-compatible, allogeneic lymphocytes greater than the number oflymphocytes administered in step (a); (2) administration of a number ofHLA-compatible, allogeneic lymphocytes at least as great as the numberof lymphocytes administered in step (a), accompanied by in vivoadministration of at least one T-cell activator to said patient; (3)administration of HLA-compatible, allogeneic ADL to said patient; and(4) administration of HLA-compatible, allogeneic ADL, accompanied by invivo administration of at least one T-cell activator to said patient;wherein more than one of said procedures is performed if no orinsufficient graft-versus-malignant cell response develops in saidpatient following said first or subsequent procedure.
 12. The method ofclaim 11, wherein step (a) of said regimen further comprises in vivoadministration of at least one T-cell activator to said patient.
 13. Themethod of claim 8, wherein said regimen comprises the following steps insequence:a) administering to said patient about 10⁷ cells/kg to about10⁹ cells/kg of HLA-compatible, allogeneic lymphocytes and at least oneT-cell activator to said patient; b) monitoring said patient for signsof mild graft-versus-host response; c) if no or insufficientgraft-versus-host response develops in said patient, administering about10⁷ cells/kg to about 10⁹ cells/kg of HLA-compatible, allogeneic ADL andat least one T-cell activator to said patient; and d) monitoring saidpatient for signs of a mild graft-versus-host response.
 14. The methodof claim 8, wherein said regimen comprises the following steps insequence:a) administering to said patient about 10⁵ cells/kg to about10⁹ cells/kg of HLA-compatible, allogeneic lymphocytes comprising ADL,and at least one T-cell activator to said patient; b) monitoring saidpatient for signs of mild graft-versus-host response; c) if no orinsufficient graft-versus-host response develops in said patient,administering about 10⁵ cells/kg to about 10⁹ cells/kg ofHLA-compatible, allogeneic ADL and at least one T-cell activator to saidpatient; and d) monitoring said patient for signs of a mildgraft-versus-host response.
 15. The method of claim 11, wherein saidT-cell activator comprises at least one T-cell signal transductionpathway activator.
 16. The method of claim 15, wherein said T-cellsignal transduction pathway activator is selected from the groupconsisting of IL1, IL2, IL4, IL5, IL6, IL7, IL12, IL13, IFNα, IFNγ,TNFα, anti-CD3, anti-CD28, phytohemagglutinin, concanavalin-A, andphorbol esters.
 17. The method of claim 13, wherein said T-cellactivator comprises at least one T-cell signal transduction pathwayactivator.
 18. The method of claim 14, wherein said T-cell activatorcomprises at least one T-cell signal transduction pathway activator. 19.The method of claim 1, wherein said allogeneic lymphocytes are selectedto have a substantially diminished graft-versus-host activity comparedto unselected lymphocytes.
 20. The method of claim 21, wherein saidselected lymphocytes are CD8+ lymphocytes.
 21. The method of claim 19,wherein said selected lymphocytes are HLA-mismatched with said patient.22. The method of claim 19 wherein said breast cancer cells aremetastatic breast cancer cells.